Sustained release microspheres

ABSTRACT

Methods for forming sustained release microspheres and the products produced thereby are provided. The microspheres have a smooth surface that includes a plurality of channel openings that are less than 1000 angstroms in diameter.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/420,361, filed on Oct. 18, 1999, now pending.

FIELD OF THE INVENTION

[0002] This invention relates to methods and compositions for formingand using sustained release microspheres. The microspheres are porousand include a plurality of channel openings that are less than 1000angstroms in diameter.

BACKGROUND OF THE INVENTION

[0003] Microparticles, microspheres, and microcapsules, referred toherein collectively as “microparticles”, are solid or semi-solidparticles having a diameter of less than one millimeter, more preferablyless than 100 microns, which can be formed of a variety of materials,including synthetic polymers, proteins, and polysaccharides.Microparticles have been used in many different applications, primarilyseparations, diagnostics, and drug delivery.

[0004] The most well known examples of microparticles used inseparations techniques are those which are formed of polymers of eithersynthetic or protein origin, such as polyacrylamide, hydroxyapatite oragarose. These polymeric microparticles are commonly used to separatemolecules such as proteins based on molecular weight and/or ionic chargeor by interaction with molecules chemically coupled to themicroparticles.

[0005] In the diagnostic area, microparticles are frequently used toimmobilize an enzyme, substrate for an enzyme, or labeled antibody,which is then interacted with a molecule to be detected, either directlyor indirectly.

[0006] In the controlled drug delivery area, molecules are encapsulatedwithin microparticles or incorporated into a monolithic matrix, forsubsequent release. A number of different techniques are routinely usedto make these microparticles from synthetic polymers, natural polymers,proteins and polysaccharides, including phase separation, solventevaporation, emulsification, and spray drying. Generally the polymersform the supporting structure of these microspheres, and the drug ofinterest is incorporated into the polymer structure. Exemplary polymersused for the formation of microspheres include homopolymers andcopolymers of lactic acid and glycolic acid (PLGA) as described in U.S.Pat. No. 5,213,812 to Ruiz, U.S. Pat. No. 5,417,986 to Reid et al., U.S.Pat. No. 4,530,840 to Tice et al., U.S. Pat. No. 4,897,268 to Tice etal., U.S. Pat. No. 5,075,109 to Tice et al., U.S. Pat. No. 5,102,872 toSingh et al., U.S. Pat. No. 5,384,133 to Boyes et al., U.S. Pat. No.5,360,610 to Tice et al., and European Patent Application PublicationNumber 248,531 to Southern Research Institute; block copolymers such astetronic 908 and poloxamer 407 as described in U.S. Pat. No. 4,904,479to Illum; and polyphosphazenes as described in U.S. Pat. No. 5,149,543to Cohen et al. Microspheres produced using polymers such as theseexhibit a poor loading efficiency and are often only able to incorporatea small percentage of the drug of interest into the polymer structure.Therefore, substantial quantities of microspheres often must beadministered to achieve a therapeutic effect.

[0007] Spherical beads or particles have been commercially available asa tool for biochemists for many years. For example, antibodiesconjugated to beads create relatively large particles specific forparticular ligands. The large antibody-coated particles are routinelyused to crosslink receptors on the surface of a cell for cellularactivation, are bound to a solid phase for immunoaffinity purification,and may be used to deliver a therapeutic agent that is slowly releasedover time, using tissue or tumor-specific antibodies conjugated to theparticles to target the agent to the desired site.

[0008] The most common method of covalently binding an antibody to asolid phase matrix is to derivative a bead with a chemical conjugationagent and then bind the antibody to the activated bead. The use of asynthetic polymeric bead rather than a protein molecule allows the useof much harsher derivatization conditions than many proteins cansustain, is relatively inexpensive, and often yields a linkage that isstable to a wide range of denaturing conditions. A number of derivatizedbeads are commercially available, all with various constituents andsizes. Beads formed from synthetic polymers such as polyacrylamide,polyacrylate, polystyrene, or latex are commercially available fromnumerous sources such as Bio-Rad Laboratories (Richmond, Calif.) and LKBProdukter (Stockholm, Sweden). Beads formed from natural macromoleculesand particles such as agarose, crosslinked agarose, globulin,deoxyribose nucleic acid, and liposomes are commercially available fromsources such as Bio-Rad Laboratories, Pharmacia (Piscataway, N.J.), andIBF (France). Beads formed from copolymers of polyacrylamide and agaroseare commercially available from sources such as IBF and Pharmacia.Magnetic beads are commercially available from sources such as DynalInc. (Great Neck, N.Y.).

[0009] One disadvantage of the microparticles or beads currentlyavailable is that they are difficult and expensive to produce.Microparticles produced by these known methods have a wide particle sizedistribution, often lack uniformity, and fail to exhibit long termrelease kinetics when the concentration of active ingredients is high.Furthermore, the polymers used in these known methods are dissolved inorganic solvents in order to form microspheres. The microspheres musttherefore be produced in special facilities designed to handle organicsolvents. These organic solvents could denature proteins or peptidescontained in the microparticles. Residual organic solvents could betoxic when administered to humans or animals.

[0010] In addition, the available microparticles are rarely of a sizesufficiently small to fit through the aperture of the size of needlecommonly used to administer therapeutics or to be useful foradministration by inhalation. For example, microparticles prepared usingpolylactic glycolic acid (PLGA) are large and have a tendency toaggregate. A size selection step, resulting in product loss, isnecessary to remove particles too large for injection. PLGA particlesthat are of a suitable size for injection must be administered through alarge gauge needle to accommodate the large particle size, often causingdiscomfort for the patient.

[0011] Generally all currently available microspheres are activated torelease their contents in aqueous media and therefore must belyophilized to prevent premature release. In addition, particles such asthose prepared using the PLGA system exhibit release kinetics based onboth erosion and diffusion. In this type of system, an initial burst orrapid release of drug is observed. This burst effect can result inunwanted side effects in patients to whom the particles have beenadministered.

[0012] Microparticles prepared using lipids to encapsulate target drugsare currently available. For example, lipids arranged in bilayermembranes surrounding multiple aqueous compartments to form particlesmay be used to encapsulate water soluble drugs for subsequent deliveryas described in U.S. Pat. No. 5,422,120 to Sinil Kim. These particlesare generally greater than 10 μm in size and are designed for intraarticular, intrathecal, subcutaneous and epidural administration.Alternatively, liposomes have been used for intravenous delivery ofsmall molecules. Liposomes are spherical particles composed of a singleor multiple phospholipid and cholesterol bilayers. Liposomes are 30 μmor greater in size and may carry a variety of water-soluble orlipid-soluble drugs. Liposome technology has been hindered by problemsincluding purity of lipid components, possible toxicity, vesicleheterogeneity and stability, excessive uptake and manufacturing orshelf-life difficulties.

[0013] Therefore, there is an on-going need for development of newmethods for making microparticles, particularly those that can beadapted for use in the separations, diagnostic and drug delivery area.Preferably, such improved microparticles would permit the sustainedrelease of active agents in a predictable, consistent manner.

SUMMARY OF THE INVENTION

[0014] The invention solves these and other problems by providingmethods and compositions for the sustained release of therapeutic and/ordiagnostic agents in vivo and in vitro. As subsequently used herein theterm “therapeutic agent” is intended to be inclusive of clinical agentswhich can be administered in microcapsular form, whether used primarilyfor treatment or diagnosis.

[0015] According to one aspect of the invention, a microsphere having asmooth surface which includes a plurality of channel openings isprovided. The channel openings have a diameter which is less than 1000angstroms as determined by gas adsorption technique for pore sizing. Themicrospheres of the invention include a macromolecule, preferably aprotein or a nucleic acid, and at least one water soluble polymer. Ingeneral, the microspheres are formed by contacting the macromolecule andat least one water soluble polymer under aqueous conditions to form themicrospheres, and the microspheres are then stabilized by eitherchemical crosslinking or exposing the microspheres to an energy source,preferably heat, or both, at a temperature which results in microsphereswhich are resistant to physical and chemical treatments such assonication and caustic solutions under these conditions. Although notwishing to be bound to any particular theory or mechanism, it isbelieved that the microspheres form during the mixing step; however,such initially formed microspheres are transient and require a furtherstep (e.g., including a crosslinking agent in the mixture and/or byapplying heat or other energy source) to stabilize thetransiently-formed microspheres. The particular conditions for formingrepresentative microspheres of the invention are described in theExamples. In the preferred embodiments, the formation reaction isconducted in the absence of the addition of oils or organic solvents.Oil is defined as a substance that is liquid fat which is insoluble inwater.

[0016] The protein component of the microsphere may be a carrier proteinor a therapeutic protein (see, e.g., Table 1). As used herein, a“carrier protein” refers to a protein which has a molecular weight of atleast about 1500 and which exists as a three dimensional structure. Thecarrier protein can also be a therapeutic protein, i.e., a protein whichhas a therapeutic activity; however, in general, the phrase “carrierprotein” will be used in this application to refer to a protein whichhas a primary function to provide a three dimensional structure for thepurpose of microsphere formation, even if the carrier protein also mayhave a secondary function as a therapeutic agent. In certain preferredembodiments, the carrier protein is an albumin, particularly, humanserum albumin. The protein microspheres of the invention, optionally,further include a therapeutic agent such as a steroid (e.g., estradiol,testosterone, prednisolone, dexamethasone, hydrocortisone, lidocainebase, procaine base), or any other such chemical entity known to bind tothe protein, preferably albumin, such as GCSF, or paclitaxel. In yetother embodiments (discussed below), the microspheres of the inventionfurther include a complexing agent and, more preferably, a therapeuticagent (preferably a peptide) which is associated with the complexingagent via an ionic or nonionic interaction. In certain otherembodiments, the protein that comprises the matrix is a therapeuticprotein (e.g., a hormone such as insulin or human growth hormone) andthe microsphere is constructed and arranged to provide sustained releaseof the therapeutic protein in vivo. More preferably, the microsphere isconstructed and arranged to provide sustained release of the therapeuticagent in the absence of significant swelling of the microsphere.

[0017] Surprisingly, the surface of the microsphere is different fromthe interior. Extensive water washing of freeze fractured microspheresdissolves much of the microsphere matrix material leaving a thin shell.In addition, the surface of the microsphere is smooth; the channel(pore) openings are less than 1000 angstroms in diameter as determinedby gas adsorption technique for por sizing using, e.g., BET technologyfor data analysis.

[0018] In general, the microspheres of the invention are formed bymixing the macromolecule, preferably a protein or a nucleic acid,together with at least one water polymer under conditions which permitthe water soluble polymer to remove water from (“dehydrate”) themacromolecule within specified or preferred ratios of macromolecule towater soluble polymer. As used herein, a “water soluble polymer” of theinvention refers to a polymer or mixture of polymers which is capable ofremoving water from or dehydrating the macromolecule or otherwisecapable of causing volume exclusion. Thus, the preferred processinvolves volume exclusion using an entirely aqueous system with no oilor organic solvents involved.

[0019] Suitable water soluble polymers include soluble linear orbranched polymers, preferably those having a high molecular weight.Polymers can be highly water soluble, moderately-water soluble, orslightly water soluble (greater than 2% wt/vol water soluble). Thepreferred water soluble polymers are water soluble or soluble in a watermiscible solvent. The water soluble polymers may be solubilized by firstbeing dissolved in a water miscible solvent and then combining thepolymer solution with an aqueous solvent. In the particularly preferredembodiments, the water soluble polymers of the invention are selectedfrom the water soluble polymers identified in Table 2. In certainembodiments, the microspheres of the invention are formed of proteinsand water soluble polymers and contain from 40 to less than 100 (wt %)protein. The final microsphere product which has been stabilized using acrosslinking agent and/or exposure to an energy source such as heat doesnot swell significantly in the presence of water (i.e., it is not ahydrogel). In the particularly preferred embodiments, the water solublepolymer is a carbohydrate-based polymer. Thus, in certain preferredembodiments, the microsphere comprises: (1) a protein, preferablyalbumin; and (2) a carbohydrate-based water soluble polymer, preferablyhetastarch, wherein the protein represents at least 40% and less than100% by weight of the microsphere. Preferably, the carbohydrate-basedpolymer represents greater than 0% and less than or equal to 30% byweight of the microsphere. In these and other embodiments, themicrospheres preferably further comprise an active agent, preferably aluteinizing hormone releasing hormone or analog thereof. In general, themicrospheres of the invention, when contacted with a solution of activeagent, are capable of incorporating at least 60%, more preferably atleast 70%, at least 80%, or at least 90%, and most preferably, at least95% or at least 98% of the active agent. The active agent-containingmicrospheres optionally are further stabilized by contacting themicrospheres with the same types of crosslinking agents and using thesame types of conditions described herein for initially stabilizing themicrospheres.

[0020] According to another aspect of the invention, a microspherefurther including a complexing agent is provided. The microsphere ofthis aspect includes: (1) a macromolecule such as a protein (e.g.,albumin, as described above); (2) at least one water soluble polymer(e.g., hetastarch (hydroxyethylstarch), PEG/PVP); and (3) a complexingagent. As used herein, a complexing agent refers to a molecule which iscapable of interacting with a therapeutic agent (discussed below) tofacilitate loading, retaining and/or otherwise delaying the release ofthe therapeutic agent from the microsphere (see, e.g., Table 3). As withall aspects of the invention, these microspheres have a smooth surfacewhich includes a plurality of channel openings that are less than 1000angstroms in diameter as determined by gas adsorption technique for poresizing and, preferably, do not contain detectable oil or organicsolvent.

[0021] According to a particularly preferred aspect of the invention, amicrosphere further including at least two complexing agents isprovided. The microsphere of this aspect includes: (1) a macromoleculesuch as a carrier protein (e.g., albumin, as described above); (2) atleast one water soluble polymer (e.g., hetastarch (hydroxyethylstarch),PEG/PVP); (3) a first complexing agent that is an anionicpolysaccharide; and (4) a second complexing agent that is a divalentmetal cation selected from the group consisting of calcium, magnesium,zinc, strontium, barium, manganese, and iron. A particularly preferredembodiment of this aspect of the invention is illustrated in Example 17.Calcium and magnesium are the preferred divalent metal cations.

[0022] As used herein, a complexing agent refers to a molecule which iscapable of interacting with a therapeutic agent (discussed below) tofacilitate loading, retaining and/or otherwise delaying the release ofthe therapeutic agent from the microsphere (see, e.g., Table 3). As withother aspects of the invention, these microspheres preferably have asmooth surface which includes a plurality of channel openings that areless than 1000 angstroms in diameter as determined by gas adsorptiontechnique for pore sizing and, more preferably, do not containdetectable oil or organic solvent.

[0023] A preferred method of incorporating the complexing agent(s) is tocombine the complexing agent(s) with a water soluble polymer in aqueoussolution, then add the macromolecule, and stabilize the microsphereswith heat and/or crosslinking agents.

[0024] In general, the complexing agent is an ionic complexing agent(i.e., the complexing agent is capable of an ionic interaction with atherapeutic agent (discussed below)) or a non-ionic complexing agent(i.e., the complexing agent is capable of a non-ionic (e.g.,hydrophobic, mixed ionic/nonionic) interaction with a therapeutic agentor with another complexing agent). Exemplary complexing agents areprovided in Table 3. Ionic complexing agents of the invention arefurther categorized into anionic complexing agents (i.e., complexingagents having a negative charge such as anionic polysaccharides, e.g.,dextran sulfate, galacturonic acids, alginates, mannuronic acid,guluronic acid, hyaluronic acid, chondroitin sulfates, heparin, chitin,chitosan, glycosaminoglycans, proteoglycans) and cationic complexingagents (i.e., complexing agents having a positive charge). The preferredcomplexing agents of the invention are anionic polysaccharides anddivalent metal cations selected from the group consisting of calcium andmagnesium.

[0025] In certain embodiments, the complexing agent is an anioniccomplexing agent having the structure of formula I:

POLY-[Y⁻]_(n)X⁺  I.

[0026] wherein POLY represents a principal chain of the anioniccomplexing agent which may be linear or branched;

[0027] wherein Y⁻ represents an anionic group, e.g., sulfates,carboxyls, phosphates, nitrates, carbonates and the like, that may becoupled to any one or more of the branches of the principal chain;

[0028] wherein X⁺ represents a cationic group, e.g., that is a counterion to the anionic group;

[0029] wherein n is an integer from 1 to 10,000, preferably, from 5 to100 and, more preferably, from 5 to 1000, and still more preferably,from 5 to 10,000; and

[0030] wherein when n is greater than 1, the n Y⁻ groups can be the sameor different.

[0031] In yet other embodiments of the invention the complexing agent isa cationic complexing agent having the structure of formula II:

POLY-[X⁺]_(n)Y⁻  II.

[0032] wherein POLY represents a principal chain of the cationiccomplexing agent which may be linear or branched;

[0033] wherein X⁺ represents a cationic group, e.g., an amino group,that may be coupled to any one or more of the branches of the principalchain;

[0034] wherein Y⁻ represents an anionic group, that is a counter ion tothe cationic group;

[0035] wherein n is an integer from 1 to 10000, preferably, from 5 to100 and, more preferably, from 5 to 1000, and most preferably, from 5 to10,000; and

[0036] wherein when n is greater than 1, the n X⁻ groups can be the sameor different.

[0037] According to another aspect of the invention, a microspherefurther including an active agent is provided (see, e.g., Table 4). Themicrospheres into which the active agent can be loaded may include acomplexing agent(s) to facilitate loading and/or modify the release ofthe active agent from the microsphere. Alternatively, the active agentcan be loaded into the above-described microspheres which lack acomplexing agent, e.g., the protein and/or the water soluble polymers ofthe invention can interact with the active agent to facilitate loadingand/or modify its release from the microsphere. In general, although theactive agent can be loaded into a microsphere of the invention duringpreparation of the microsphere, it is preferable to load the activeagent into a preformed microsphere of the invention and, morepreferable, to load the active agent into a preformed microsphere whichcontains a complexing agent(s) to facilitate loading and/or sustainedrelease of the agent. In contrast to hydrogel microspheres, themicrospheres of the invention do not swell significantly in water and,further, the microspheres do not require swelling in order to providesustained release of the therapeutic protein and/or physiologicallyactive agent from the microsphere.

[0038] As used herein, an active agent refers to an agent which has adiagnostic or therapeutic activity. Accordingly, an active agent caninclude a detectable label (e.g., a radioactive label) that is usefulfor identifying the locations of the released agent in vivo; Activeagents also include therapeutic agents which are useful for treating adisease or condition. In certain embodiments, the preferredphysiologically active agents are protein or peptide agents. Suchprotein or peptide agents typically can be further divided intocategories, based upon the activity of the agent or the type of diseaseor condition that is being treated. The categories of physiologicallyactive agents which can be used in the present invention include, butare not limited to, antibiotics, hematopoietics, antiinfective agents,antidementia agents, antiviral agents, antitumoral agents, antipyretics,analgesics, antiinflammatory agents, antiulcer agents, antiallergicagents, antidepressants, psychotropic agents, cardiotonics,antiarrythmic agents, vasodilators, antihypertensive agents such ashypotensive diuretics, antidiabetic agents, anticoagulants, cholesterollowering agents, therapeutic agents for osteoporosis, hormones, vaccinesand so on (see, e.g., Table 4).

[0039] The physiologically active peptide or protein which is employedin accordance with the present invention is a peptide composed of two ormore amino acids. Preferably, such a peptide has a molecular weightgreater than 200, e.g., in the range of about 200 to 200000. The morepreferred molecular weight range is about 200 to 100000. More specificexamples of physiologically active agents, including non-protein agents,which can be used in connection with the methods and compositions of theinvention are provided in the detailed description of the invention.

[0040] According to still another aspect of the invention, a method forforming a microsphere is provided. The method involves: (1) forming anaqueous mixture containing a macromolecule, preferably a protein or anucleic acid, and a water soluble polymer, preferably acarbohydrate-based polymer such as hetastarch; (2) allowing themicrospheres to form in the aqueous mixture; (3) stabilizing themicrospheres, preferably by contacting the microspheres with acrosslinking agent and/or exposing the microspheres to an energy source,preferably heat, under conditions sufficient to stabilize themicrospheres; wherein the macromolecule is present in the aqueousmixture in an amount sufficient to form a microsphere that contains atleast 40% and less than 100% by weight macromolecule. Although notwishing to be bound to any particular theory or mechanism, it isbelieved that the microspheres form during the mixing step; however,such initially formed microspheres are transient and require a furtherstep (e.g., including a crosslinking agent in the mixture and/or byapplying heat or other energy source) to stabilize thetransiently-formed microspheres. Exemplary methods of preparing themicrospheres are provided in the Examples.

[0041] According to yet another aspect of the invention, apharmaceutical composition of matter and method of making saidcomposition are provided. In certain embodiments, the compositionincludes a container containing a single dose of microspheres containingan active agent for treating a condition that is treatable by thesustained release of an active agent from the microspheres. The numberof microspheres in the single dose is dependent upon the amount ofactive agent present in each microsphere and the period of time overwhich sustained release is desired. Preferably, the single dose isselected to achieve the sustained release of the active agent over aperiod of from about 1 to about 180 days, wherein the single dose ofmicrospheres is selected to achieve the desired release profile fortreating the condition.

[0042] According to another aspect of the invention, a syringecontaining any of the microspheres disclosed herein is provided. Forexample, the composition can includes a syringe containing a single doseof microspheres containing an active agent for treating a condition thatis treatable by the sustained release of the active agent from themicrospheres. Preferably, a needle is attached to the syringe, whereinthe needle has a bore size that is from 14 to 30 gauge.

[0043] Remarkably, the microspheres of the invention can be prepared tohave a dimension which permits the delivery of microspheres using aneedleless syringe (MediJector, Derata Corporation, Minneapolis, Minn.55427), thereby eliminating the disposal problems inherent to needleswhich must be disposed as biohazard waste product. Thus, according to aparticularly preferred aspect of the invention, a needleless syringecontaining one or more doses of microspheres containing an active agentfor treating a condition is provided. The microspheres can be preparedto have qualities suitable to be delivered by other parenteral andnon-parenteral routes such as oral, buccal, intrathecal, nasal,pulmonary, transdermal, transmucosal and the like.

[0044] According to still other embodiments of the invention, nucleicacid-containing microspheres are provided. The nucleic acid-containingmicrospheres include: (1) a nucleic acid (e.g., plasmid, viral vector,oligonucleotide, RNA, antisense and missense nucleic acids); (2) apolycationic polymer (e.g., polylysine); and (3) a water soluble polymer(as described above). Thus, a method for forming the nucleicacid-containing microspheres is provided. The method involves: (1)combining, in one or more aqueous solutions, a nucleic acid, apolycationic polymer and a water soluble polymer to form an aqueousmixture which can be a mono- or multi-phase; and (2) subjecting theaqueous mixture to a crosslinking agent and/or an energy source underconditions (e.g., of concentration, incubation time) sufficient tostabilize a microsphere. Exemplary methods of forming the nucleic acidmicrospheres are provided in the Examples.

[0045] These and other aspects of the invention will be described ingreater detail below. Throughout this disclosure, all technical andscientific terms have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains unlessdefined otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1 is a graph showing the cumulative percent of radiolabeledpolyethylene glycol (PEG) and radiolabeled bovine serum albumin (BSA)released from microspheres versus the square root of time in hours. Theblack square symbol represents PEG, and the open square symbolrepresents BSA.

[0047]FIG. 2 is a graph showing the cumulative percent of radiolabeledbovine serum albumin (BSA) released from microspheres prepared withthree different concentrations of polymer versus the square root of timein hours. The gray square symbol represents a total polymerconcentration of 50%, the open square symbol represents a total polymerconcentration of 40%, and the black triangle symbol represents a totalpolymer concentration of 25%.

[0048]FIG. 3 is a graph showing the cumulative percent of radiolabeledpolyethylene glycol (PEG) released from microspheres prepared with threedifferent concentrations of polymer versus the square root of time inhours. The open triangle symbol represents a total polymer concentrationof 50%, the black square symbol represents a total polymer concentrationof 40%, and the open square symbol represents a total polymerconcentration of 25%.

[0049]FIG. 4 is a graph showing the cumulative percent of radiolabeledbovine serum albumin (BSA) released from microspheres prepared withthree different concentrations of polymer at various incubationtemperatures versus the square root of time in hours. The open squaresymbol represents a total polymer concentration of 25% and incubation at58° C., the black square symbol represents a total polymer concentrationof 25% and incubation at 70° C., the open circle symbol represents atotal polymer concentration of 40% and incubation at 58° C., the blackcircle symbol represents a total polymer concentration of 40% andincubation at 70° C., the open triangle symbol represents a totalpolymer concentration of 50% and incubation at 58° C., the blacktriangle symbol represents a total polymer concentration of 50% andincubation at 70° C., the light “X” symbol represents a total polymerconcentration of 25% and incubation at 37° C. and 58° C., and the dark“X” symbol represents a total polymer concentration of 25% andincubation at 37° C. and 70° C.

[0050]FIG. 5 is a graph showing the cumulative percent of radiolabeledpolyethylene glycol (PEG) released from microspheres prepared with threedifferent concentrations of polymer at various incubation temperaturesversus the square root of time in hours. The symbols are the same asthose described in FIG. 4.

[0051]FIG. 6 is a graph showing the cumulative percent of radiolabeledpolyethylene glycol (PEG) released from microspheres prepared with threedifferent concentrations of polymer at an incubation temperatureincluding 58° C. versus the square root of time in hours. The opentriangle symbol represents a total polymer concentration of 50% andincubation at 58° C., the black square symbol represents a total polymerconcentration of 40% and incubation at 58° C., the gray triangle symbolrepresents a total polymer concentration of 25% and incubation at 37° C.and 58° C., and the open square symbol represents a total polymerconcentration of 25% and incubation at 58° C.

[0052]FIG. 7 is a bar graph showing the amount of expressed gene productby beta-galactosidase activity in milliunits versus microsphereformation for naked DNA, cationic liposomes containing DNA, and DNAmicrospheres.

[0053]FIG. 8 is a graph of cumulative percent release of leuprolideacetate from microspheres over time in days.

[0054]FIG. 9 is a graph of cumulative percent nafarelin acetate releaseversus time in hours for three concentrations of zinc sulfate usedduring microsphere preparation. The circle symbol represents 0.01M zincsulfate; the square symbol represents 0.1M zinc sulfate; and thetriangle symbol represents 1M zinc sulfate.

[0055]FIG. 10 shows the sustained release of estradiol in vitro from themicrospheres of the invention.

[0056]FIG. 11 shows the sustained release of estradiol in vivo (serumconcentrations) from the microspheres of the invention.

[0057]FIG. 12 shows the efficiency of diclofenac Na incorporation byadjusting the incubation conditions.

[0058]FIG. 13 shows a representative size distribution profile for themicrospheres of the invention.

DESCRIPTION OF THE INVENTION

[0059] The invention provides methods and compositions for the sustainedrelease of therapeutic and/or diagnostic agents in vivo and/or in vitro.The microspheres have a generally uniform size and shape, ranging insize from about 0.5 microns to about 20 microns, depending upon thefabrication conditions. The characteristics of the microspheres may bealtered during preparation by manipulating the water soluble polymerconcentration, reaction temperature, pH, protein concentration,crosslinking agent, and/or the length of time the macromolecule isexposed to the crosslinking agent and/or the energy source.

[0060] The microspheres are useful for a wide variety of separations,diagnostic, therapeutic, industrial, commercial, cosmetic, and researchpurposes or for any purpose requiring the incorporation of andstabilization of an active molecule, reactant or drug. Thus, themicrospheres of the invention are useful for medical and diagnosticapplications, such as drug delivery, vaccination, gene therapy andhistopathological or in vivo tissue or tumor imaging. Accordingly, themicrospheres are suitable for oral or parenteral administration; mucosaladministration; ophthalmic administration; intravenous, subcutaneous,intra articular, or intramuscular injection; administration byinhalation; and topical administration.

[0061] According to one aspect of the invention, a microsphere having asmooth surface which includes a plurality of channel openings isprovided. Each channel opening has a diameter which is less than 1000angstroms as determined by gas adsorption technique for pore sizingusing, e.g., BET methodology for data analysis.

[0062] The microspheres are prepared by mixing or dissolvingmacromolecules with a water soluble polymer or mixture of water solublepolymers, such as linear or branched polymers of Table 2, at a pH nearthe isoelectric point of the macromolecule. The macromolecule andpolymer mixture is exposed to a crosslinking agent and/or an energysource, such as heat, under conditions sufficient to stabilize themicrospheres. The microspheres are then separated from theunincorporated reagents by separation methods such as filtration orcentrifugation.

[0063] In general, the macromolecule or combination of macromoleculescompose at least 40% and less than 100% by weight of the final weight ofeach microsphere. Preferably, the polymer concentration in themicrosphere is greater than 0% and less than or equal to 30% by weight.The types of macromolecules forming the microspheres include, but arenot limited to, proteins, peptides, carbohydrates, nucleic acids,viruses, or mixtures thereof.

[0064] Each microsphere is composed of macromolecules and polymermolecules, which are intertwined or interspersed in the microsphere andare generally homogeneously distributed. Although not wishing to bebound to any particular theory or mechanism, it is believed that theinner matrix is water soluble, and, when solubilized, the inner matrixdiffuses through the outer surface under appropriate conditions asexplained in more detail below. The microspheres exhibit a narrow sizedistribution and have a generally uniform shape. Size distribution isalso adjustable by modifying the conditions and reagents used during thepreparation process and may be associated with release kinetics asdescribed below. The microspheres are generally less than 10 μm indiameter. See FIG. 13 for a representative size distribution of themicrospheres of the invention. The uniform shape of the microspheres issubstantially spherical, which is why the microparticles are alsoreferred to herein as “microspheres”.

[0065] The outer surface of each microsphere is permeable to water anddissolved macromolecules and not only allows aqueous fluids to enter themicrosphere, but also allows solubilized macromolecule and polymer toexit the microsphere. The microspheres can be made to releasemacromolecule and polymer from the interior of the microsphere whenplaced in an appropriate aqueous medium, such as body fluids or aphysiologically acceptable buffer under physiological conditions over aprolonged period of time, thereby providing sustained release ofmacromolecules. In addition, the microspheres can be made to releasemacromolecule without an initial burst or rapid release ofmacromolecule. Sustained release is defined herein as release ofmacromolecules over an extended period of time. The amount of time overwhich the macromolecules continue to be released from the microspheredepends on the characteristics of the macromolecule being released andthe parameters used to form the microspheres, but in all cases is longerthan that of free aqueous diffusion of the macromolecule. Microspherescontaining pharmaceutical compounds can be made to release thepharmaceutical compound with the macromolecule and polymer as describedabove.

[0066] As discussed briefly above and in more detail below, thecharacteristics of the microspheres may be manipulated duringpreparation by adjusting the type of polymer, polymer concentration,polymer composition, incubation temperature, pH, macromoleculeconcentration, or the length of time the macromolecule is exposed to theenergy source.

[0067] The microspheres may be administered to a human or animal by oralor parenteral administration, including intravenous, subcutaneous orintramuscular injection; administration by inhalation; intra articularadministration; mucosal administration; ophthalmic administration; andtopical administration. Intravenous administration includescatheterization or angioplasty. Administration may be for purposes suchas therapeutic and diagnostic purposes as discussed below.

[0068] Formation of Microspheres

[0069] Microspheres are produced by mixing macromolecules in an aqueousmixture with a water soluble polymer or mixture of polymers to form themicrospheres and optionally, thereafter contacting the microspheres witha crosslinking agent and/or an energy source, preferably heat, underconditions sufficient to stabilize the microspheres. The solution ispreferably an aqueous solution. Either the macromolecule solution isadded to the polymer or the polymer solution is added to themacromolecule solution to cause removal of water from, or dehydrationof, the macromolecules. This process is also referred to by thoseskilled in the art as volume exclusion. Although not wishing to be boundto any particular theory or mechanism, it is believed that themicrospheres form during the mixing step; however, such initially formedmicrospheres are transient and require a further step (e.g., including acrosslinking agent in the mixture and/or by applying heat or otherenergy source) to stabilize the transiently-formed microspheres.

[0070] The pH of the macromolecule-polymer solution is adjusted, eitherbefore, after or during the mixing of the polymer with themacromolecule, to a pH near the isoelectric point (pI) of themacromolecule, preferably within 3 to 4 pH units of the pI of themacromolecule, most preferably within 1.5 to 2 pH units of the pl of themacromolecule.

[0071] The pH adjustment may be made by adding an acid, base, either insolution or solid form, or a buffer or other pH-adjusting solution orsalt, to either the macromolecule solution, the polymer solution, or tothe mixture of macromolecule and polymer in accordance with methods wellknown to those skilled in the art. Preferably the polymer is dissolvedin a buffer having a pH near the pI of the macromolecule, and then thepH-adjusted polymer solution is added to the macromolecule, which hasbeen dissolved in an aqueous solution. The pH of the final solutionshould remain near the pI of the macromolecule.

[0072] The macromolecule and polymer solution is then exposed to acrosslinking agent and/or an energy source, such as heat, radiation, orionization, alone or in combination with sonication, vortexing, mixingor stirring, for a predetermined length of time to stabilize themicrospheres. The resulting microspheres are then separated from anyunincorporated components present in the solution by, physicalseparation methods well known to those skilled in the art and may thenbe washed.

[0073] The length of incubation time is dependent upon the respectiveconcentrations of polymer and macromolecule and the level of energy ofthe energy source. Microsphere stabilization can begin to occurimmediately upon exposure to the energy source. Preferably, themacromolecule and polymer mixture is heated at a temperature greaterthan room temperature for between approximately 5 minutes and 24 hours.Most preferably, the polymer and macromolecules are mixed, by stirringor rocking, for 30 minutes at a temperature between approximately 37° C.and 70° C.

[0074] Macromolecule

[0075] The macromolecule forming the microsphere is any molecule havinga tertiary and quaternary structure or capable of having a tertiary andquaternary structure. Most preferably, the macromolecule is abiomolecule such as a protein, including enzymes and recombinantproteins, a peptide, carbohydrate, polysaccharide, carbohydrate- orpolysaccharide-protein conjugate, nucleic acid, virus, virus particle,conjugate of a small molecule (such as a hapten) and protein, ormixtures thereof. An organic or inorganic natural or syntheticpharmaceutical compound or drug may be incorporated into themicrospheres by attaching the drug to a macromolecule, such as aprotein, and then forming the microspheres from the macromolecule-drugcomplex or conjugate. It will be understood by those skilled in the artthat a compound incapable of having a tertiary and quaternary structurecan be formed into a microsphere by incorporation or coupling of thecompound into a carrier molecule that has a tertiary and quaternarystructure. It will be further understood by those skilled in the artthat the macromolecule can be a portion of a molecule such as, forexample, a peptide, a single-stranded segment of a double-strandednucleic acid molecule, or a virus particle, having a tertiary andquaternary structure. It will also be understood that the term“macromolecule” includes a plurality of macromolecules and includescombinations of different macromolecules such as a combination of apharmaceutical compound and an affinity molecule for targeting thepharmaceutical compound to a tissue, organ or tumor requiring treatment.It will be further understood that an affinity molecule can be eitherthe receptor portion or the ligand portion of a receptor-ligandinteraction. Examples of ligands that interact with other biomoleculesinclude viruses, bacteria, polysaccharides, or toxins that act asantigens to generate an immune response when administered to an animaland cause the production of antibodies.

[0076] Suitable compounds or macromolecules include, but are not limitedto, betaxolol™, diclofenac™, doxorubicin, rifampin™, leuprolide acetate,luteinizing hormone releasing hormone (LHRH), (D-Tryp6)-LHRH, nafarelinacetate, insulin, sodium insulin, zinc insulin, protamine, lysozyme,alpha-lactalbumin, basic fibroblast growth factor (bFGF),beta-lactoglobulin, trypsin, carbonic anhydrase, ovalbumin, bovine serumalbumin (BSA), human serum albumin (HSA), phosphorylase b, alkalinephosphatase, beta-galactosidase, IgG, fibrinogen, poly-L-lysine, IgM,DNA, desmopressin acetate™, growth hormone releasing factor (GHRF),somatostatin, antide, Factor VIII, G-CSF/GM-CSF, human growth hormone(hGH), beta interferon, antithrombin III, alpha interferon, alphainterferon 2b.

[0077] The incubation conditions are typically optimized to incorporateapproximately 100% of the macromolecule in the reaction mixture byadjusting the pH, temperature, concentration of macromolecule, or lengthof reaction or incubation. In general, less energy is required to formmicrospheres at higher concentrations of macromolecule.

[0078] Microspheres composed of nucleic acids are preferably prepared byfirst mixing the nucleic acid either with a protein, such as bovineserum albumin, or, because nucleic acids are anions, the addition of acation, such as polylysine, which aids greatly in the formation ofmicrospheres.

[0079] As mentioned above, a small molecule or compound incapable ofhaving a tertiary and quaternary structure, such as a peptide orpharmaceutical compound, can be formed into a microsphere byincorporation or coupling of the compound into a carrier molecule thathas a tertiary and quaternary structure. This may be achieved in severalways. For example, microspheres may be formed as described herein usinga macromolecule having a tertiary and quaternary structure, such as aprotein, and then the small molecule or compound is bound inside and/oron the surface of the microsphere. Alternatively, the small molecule orcompound is bound to the macromolecule having a tertiary and quaternarystructure using hydrophobic or ionic interactions and then microspheresare formed from the macromolecule-small molecule complex using themethod described herein. A third way to make microspheres from smallmolecules is to prepare microspheres using a macromolecule having atertiary and quaternary structure in such a way that the microsphere hasa net charge and then add a small molecule or compound having anopposite net charge so that the small molecule is physically attractedto and remains attached to the microsphere, but can be released overtime under the appropriate conditions. Alternatively, different types ofnon-covalent interactions such as hydrophobic or affinity interactionsmay be used to allow attachment and subsequent release of smallmolecules.

[0080] When preparing microspheres containing protein, a proteinstabilizer such as glycerol, fatty acids, sugars such as sucrose, ionssuch as zinc, sodium chloride, or any other protein stabilizers known tothose skilled in the art may be added prior to the addition of thepolymers during microsphere formation to minimize protein denaturation.

[0081] Labeled Macromolecule

[0082] Prior to being incorporated into a microsphere, the macromoleculemay be labeled with a detectable label. The various types of labels andmethods of labeling proteins and nucleic acid molecules are well knownto those skilled in the art. It will be understood by those skilled inthe art that a magnetic substance, such as a metal, is included withinthe definition of the term label. For example, the macromolecule can belabeled with a metallic substance, such as a metal, so that themicrospheres can be separated from other substances in a solution withthe aid of a magnetic device.

[0083] Several other specific labels or reporter groups are set forthbelow. For example, the label can be a radiolabel such as, but notrestricted to, [32]P, [3]H, [14]C, [35]S, [125]I, or [131]I. A [32]Plabel can be conjugated to a protein with a conjugating reagent orincorporated into the sequence of a nucleic acid molecule bynick-translation, end-labeling or incorporation of labeled nucleotide.For example, a [3]H, [14]C or [35]S label can be incorporated into anucleotide sequence by incorporation of a labeled precursor or bychemical modification, whereas an [125]I or [131]I label is generallyincorporated into a nucleotide sequence by chemical modification.Detection of a label can be by methods such as scintillation counting,gamma ray spectrometry or autoradiography.

[0084] The label can also be a Mass or Nuclear Magnetic Resonance (NMR)label such as, for example, [13]C, [15]N, or [19]O. Detection of such alabel can be by Mass Spectrometry or NMR. Dyes, chemiluminescent agents,bioluminescent agents and fluorogens can also be used to label themacromolecule. Examples of dyes useful for labeling nucleic acidsinclude ethidium bromide, acridine, propidium and other intercalatingdyes, and 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma Chemical Company,St. Louis, Mo.) or other nucleic acid stains. Examples of fluorogensinclude fluorescein and derivatives, phycoerythrin, allo-phycocyanin,phycocyanin, rhodamine, Texas Red or other fluorogens. The fluorogensare generally attached by chemical modification. The dye labels can bedetected by a spectrophotometer and the fluorogens can be detected by afluorescence detector.

[0085] The macromolecule can also be labeled with a chromogen (enzymesubstrate) to provide an enzyme or affinity label, or enzyme.Alternatively, the macromolecule can be biotinylated so that it can beutilized in a biotin-avidin reaction, which may also be coupled to alabel such as an enzyme or fluorogen. The macromolecule can be labeledwith peroxidase, alkaline phosphatase or other enzymes giving achromogenic or fluorogenic reaction upon addition of substrate.

[0086] A label can also be made by incorporating any modified base,amino acid, or precursor containing any label, incorporation of amodified base or amino acid containing a chemical group recognizable byspecific antibodies, or by detecting any bound antibody complex byvarious means including immunofluorescence or immuno-enzymaticreactions. Such labels can be detected using enzyme-linked immunoassays(ELISA) or by detecting a color change with the aid of aspectrophotometer.

[0087] Coated Microspheres

[0088] Molecules, distinct from the macromolecules of which themicrospheres are composed, may be attached to the outer surface of themicrospheres by methods known to those skilled in the art to “coat” or“decorate” the microspheres. The ability to attach molecules to theouter surface of the microsphere is due to the high concentration ofmacromolecule in the microsphere. These molecules are attached forpurposes such as to facilitate targeting, enhance receptor mediation,and provide escape from endocytosis or destruction. For example,biomolecules such as phospholipids may be attached to the surface of themicrosphere to prevent endocytosis by endosomes; receptors, antibodiesor hormones may be attached to the surface to promote or facilitatetargeting of the microsphere to the desired organ, tissue or cells ofthe body; and polysaccharides, such as glucans, may be attached to theouter surface of the microsphere to enhance or to avoid uptake bymacrophages.

[0089] In addition, one or more cleavable molecules may be attached tothe outer surface of or within the microspheres. The cleavable moleculesare designed so that the microspheres are first targeted to apredetermined site under appropriate biological conditions and then,upon exposure to a change in the biological conditions, such as a pHchange, the molecules are cleaved causing release of the microspherefrom the target site. In this way, microspheres are attached to or takenup by cells due to the presence of the molecules attached to the surfaceof the microspheres. When the molecule is cleaved, the microspheresremain in the desired location, such as within the cytoplasm or nucleusof a cell, and are free to release the macromolecules of which themicrospheres are composed. This is particularly useful for drugdelivery, wherein the microspheres contain a drug that is targeted to aspecific site requiring treatment, and the drug can be slowly releasedat that site. The preferred site of cleavage is a diester bond.

[0090] The microspheres may also be coated with one or more stabilizingsubstances, which may be particularly useful for long term depoting withparenteral administration or for oral delivery by allowing passage ofthe microspheres through the stomach or gut without dissolution. Forexample, microspheres intended for oral delivery may be stabilized witha coating of a substance such as mucin, a secretion containingmucopolysaccharides produced by the goblet cells of the intestine, thesubmaxillary glands, and other mucous glandular cells.

[0091] Additionally, the particles can be non-covalently coated withcompounds such as fatty acids or lipids. The coating may be applied tothe microspheres by immersion in the solubilized coating substance,spraying the microspheres with the substance or other methods well knownto those skilled in the art.

[0092] In certain of the preferred embodiments, the microspheres of theinvention include a protein and at least one water soluble polymer. Asdiscussed above, the microspheres are formed by contacting the proteinand at least one water soluble polymer under aqueous conditions to formthe microspheres, and the microspheres are then stabilized by eitherchemical crosslinking or exposing the microspheres to an energy source,preferably heat, or both, under conditions (e.g., concentration,temperature) which result in microspheres which are resistant tophysical and chemical treatments such as sonication and causticsolutions. The particular conditions for forming representativemicrospheres of the invention are described in the Examples. In thepreferred embodiments, the formation reaction is conducted in theabsence of the addition of oils or organic solvents.

[0093] The protein component of the microsphere may be a carrier proteinor a therapeutic protein (see, e.g., Table 1), which represents fromabout 40 to less than 100% (wt %) of the microsphere.

[0094] As used herein, a “carrier protein” refers to a protein which hasa molecular weight of at least about 1500 and which exists as a threedimensional structure. The carrier protein can also be a therapeuticprotein, i.e., a protein which has a therapeutic activity; however, ingeneral, the phrase “carrier protein” will be used in this applicationto refer to a protein which has a primary function to provide a threedimensional structure, for the purpose of microsphere formation, even ifthe carrier protein also may have a secondary function as a therapeuticagent. In certain preferred embodiments, the carrier protein is albumin,particularly, human serum albumin. The protein microspheres of theinvention, optionally, further include a therapeutic agent such as asteroid (e.g., estradiol, testosterone, prednisolone, dexamethasone,hydrocortisone, lidocaine base, procaine base), or any other suchchemical entity known to bind to albumin such as GCSF, or paclitaxel. Inyet other embodiments (discussed below), the microspheres of theinvention further include a complexing agent (preferably, an ioniccomplexing agent) and, more preferably, a therapeutic agent (preferablya peptide) which is associated with the complexing agent via an ionic ornon-ionic interaction. In certain other embodiments, the protein thatcomprises the matrix is a therapeutic protein (e.g., a hormone such asinsulin or human growth hormone) and the microsphere is constructed andarranged to provide sustained release of the therapeutic protein invivo. More preferably, the microsphere is constructed and arranged toprovide sustained release of the therapeutic protein in the absence ofsignificant swelling of the microsphere. TABLE 1 Proteins PROTEINSCARRIER PROTEINS OR THERAPEUTIC PROTEINS OR MOLECULES PEPTIDES ORMOLECULES Albumins (preferably, human serum Insulin; human growthhormone; albumin); HAS; BSA; IgG; IgM; GCSF; GMCSF; LHRH; VEGF; insulin;hGH; lysozyme; alpha- basic fibroblast growth factor lactoglobulin;basic fibroblast growth (bFGF); DNA; RNA; asparaginase; factor; VEGF;chymotrypsin; trypsin; tPA; urokinase; streptokinase; carbonicanhydrase; ovalbumin; interferon; glucagon; ACTH; phosphorylase b;alkaline oxytocin; secretin; vasopressin; phosphatase;beta-galactosidase; and levothyroxin. fibrinogen; poly-l-lysine; DNA;immunoglobulins (e.g., antibodies); casein; collagen; soy protein; andgelatin.

[0095] In general, the microspheres of the invention are formed bymixing the protein together with at least one water soluble polymerunder suitable conditions which, preferably, permit the water solublepolymer to remove water from (“dehydrate”) the protein (see e.g., Table2) within specified or preferred ratios (wt/wt) of protein to watersoluble polymer (e.g., ratios range from about 1 protein:1 polymer toabout 1 protein:1000 polymer). The preferred ratio of protein to watersoluble polymer in the microsphere formation reaction is in the rangefrom about 1 protein:5 polymer to about 1 protein:30 polymer. As notedabove, a “water soluble polymer” of the invention refers to a polymer ormixture of polymers which, preferably, are capable of interacting withthe macromolecule (e.g., protein or other molecule) to cause volumeexclusion. Thus, the preferred process involves using an entirelyaqueous system with no oil or organic solvents involved.

[0096] Suitable water soluble polymers include soluble linear orbranched polymers, preferably those having a high molecular weight.Polymers can be highly water soluble, moderately-water soluble, orslightly water soluble (greater than 2% wt/vol water soluble). Thepreferred water soluble polymers are water soluble or soluble in a watermiscible solvent. The water soluble polymers may be solubilized by firstbeing dissolved in a water miscible solvent and then combining thepolymer solution with an aqueous solvent. In the particularly preferredembodiments, the water soluble polymers of the invention are selectedfrom water soluble polymers identified in Table 2. In the particularlypreferred embodiments, the water soluble polymer is a carbohydrate-basedpolymer.

[0097] The preferred polymer is polyvinylpyrrolidone, polyethyleneglycol, dextran, polyoxyethylene-polyoxypropylene copolymer, polyvinylalcohol, starch, hetastarch, or mixtures thereof, the characteristics ofwhich are described in more detail below. The polymer or polymer mixturemay be prepared in accordance with the methods set forth in U.S. Pat.No. 5,525,519 to James E. Woiszwillo, or PCT Patent Application No.US93-00073 (International Publication No. WO 93/14110), filed Jan. 7,1993 and published on Jul. 22, 1993 by James E. Woiszwillo, both ofwhich are incorporated herein by reference), in which the polymer isdissolved in water or an aqueous solution, such as a buffer, in aconcentration between approximately 1 and 50 g/100 ml depending on themolecular weight of the polymer. The preferred total polymerconcentration in the polymer solution is between 10% and 80%, expressedas weight/volume percent. The preferred concentration of each polymer inthe polymer solution is between 5% and 50%. As discussed above, the pHof the polymer solution may be adjusted before being combined with themacromolecule so that the addition of the polymer causes a pH adjustmentof the macromolecule solution, most preferably within one pH unit of thepI. The pH may be adjusted during the preparation of the polymersolution by preparing the polymer in a buffer having a predetermined pH.Alternatively, the pH may be adjusted after preparation of the polymersolution with an acid or a base.

[0098] Polyoxyethylene-polyoxypropylene copolymer, also known aspoloxamer, is sold by BASF (Parsippany, N.J.) and is available in avariety of forms with different relative percentages of polyoxyethyleneand polyoxypropylene within the copolymer.

[0099] PVP is a non-ionogenic, hydrophilic polymer having a meanmolecular weight ranging from approximately 10,000 to 700,000 and thechemical formula (C₆H₉NO)[n]. PVP is also known aspoly[1-(2-oxo-1-pyrrolidinyl)ethylene], Povidone™, Polyvidone™, RP 143™,Kollidon™, Peregal ST™, Periston™, Plasdone™, Plasmosan™, Protagent™,Subtosan™, and Vinisil™. PVP is non-toxic, highly hygroscopic andreadily dissolves in water or organic solvents.

[0100] Polyethylene glycol (PEG), also known as poly(oxyethylene)glycol, is a condensation polymer of ethylene oxide and water having thegeneral chemical formula HO(CH₂CH₂O)[n]H. TABLE 2 Water Soluble PolymersWATER SOLUBLE POLYMERS 1) carbohydrate-based polymers, such asmethylcellulose, carboxymethyl cellulose-based polymers, dextran,polydextrose, derivatized chitins, chitosan, and starch (includinghetastarch), and derivatives thereof; 2) polyaliphatic alcohols such aspolyethylene oxide and derivatives thereof including polyethylene glycol(PEG), PEG-acrylates, polyethylene imine, polyvinyl acetate, andderivatives thereof; 3) poly(vinyl) polymers such as poly(vinyl)alcohol, poly(vinyl)pyrrolidone, poly(vinyl)phosphate,poly(vinyl)phosphonic acid, and derivatives thereof; 4) polyacrylicacids and derivatives thereof; 5) polyorganic acids, such as polymaleicacid, and derivatives thereof; 6) polyamino acids, such as polylysine,and polyimino acids, such as polyimino tyrosine, and derivativesthereof; 7) co-polymers and block co-polymers, such as poloxamer 407 orPluronic L-101 ™ polymer, and derivatives thereof; 8) tert-polymers andderivatives thereof; 9) polyethers, such as poly(tetramethylene etherglycol), and derivatives thereof; 10) naturally occurring polymers, suchas zein and pullulan, and derivatives thereof; 11) polyimids, such aspolyn-tris(hydroxymethyl)- methylmethacrylate, and derivatives thereof;12) surfactants, such as poly- oxyethylene sorbitan, and derivativesthereof; 13) polyesters such as poly(ethylene glycol)(n)monomethyl ethermono(succinimidylsuccinate)- ester, and derivatives thereof; 14)branched and cyclo-polymers, such as branched PEG and cyclodextrins, andderivatives thereof; and 15) polyaldehydes, such aspoly(perfluoropropylene oxide-b- perfluoroformaldehyde), and derivativesthereof.

[0101] Dextran is a term applied to polysaccharides produced by bacteriagrowing on a sucrose substrate. Native dextrans produced by bacteriasuch as Leuconostoc mesenteroides and Lactobacteria dextranicum usuallyhave a high molecular weight. Dextrans are routinely available and areused in injectable form as plasma expanders in humans.

[0102] Polyvinyl alcohol (PVA) is a polymer prepared from polyvinylacetates by replacement of the acetate groups with hydroxyl groups andhas the formula (CH₂CHOH)[n]. Most polyvinyl alcohols are soluble inwater.

[0103] PEG, dextran, PVA and PVP are commercially available fromchemical suppliers such as the Sigma Chemical Company (St. Louis, Mo.).

[0104] Most preferably, the polymer is a polymer mixture containing anaqueous solution of PVP having a molecular weight between 10,000 and360,000, most preferably 40,000, and PEG having a molecular weightbetween 200 and 35,000. PVP having a molecular weight of 40,000 and PEGhaving a molecular weight of 3500 is preferred. Preferably, the PVP isdissolved in an acetate buffer and PEG is added to the aqueous PVPsolution. The concentration of each polymer is preferably between 1 and40 g/100 ml depending of the molecular weight of each polymer. Equalconcentrations of PVP and PEG generally provide the most favorablepolymer mixture for the formation of microspheres.

[0105] An alternative preferred polymer is a dextran, having a molecularweight from approximately 3000 to 500,000 daltons.

[0106] The volume of polymer added to the macromolecule varies dependingon the size, quantity and concentration of the macromolecule.Preferably, two volumes of the polymer mixture at a 5-50% total polymerconcentration are added to one volume of a solution containing themacromolecule. The polymer is present in a liquid phase during thereaction with macromolecule.

[0107] In certain of the embodiments and, in particular, the embodimentsof the microspheres which do not further contain a complexing agent, thewater soluble polymer preferably is not PEG, PVP, dextran,nonylphenol-ethoxylates, and/or polyvinyl alcohol.

[0108] Complexing Agents

[0109] According to another aspect of the invention, a microspherefurther including a complexing agent is provided. As used herein, acomplexing agent refers to a molecule which is capable of facilitatingloading, retaining and/or otherwise delaying the release of thetherapeutic agent from the microsphere (see, e.g., Table 3). In certainembodiments, the microsphere of this aspect includes: (1) amacromolecule such as a protein (e.g., albumin, as described above); (2)at least one water soluble polymer (e.g., hetastarch, PEG/PVP, asdescribed above); and (3) a complexing agent. TABLE 3 Complexing AgentsCOMPLEXING AGENTS OTHER INTERACTION (including non-ionic and IONIC mixedionic and non- POLYANIONIC POLYCATIONIC ionic interactions) Dextransulfate Poly-lysine Albumin Heparin sulfate Poly arginine IgG Heparansulfate Poly imino acids IgM Chondroitin sulfate Poly imino tyrosinehydrophobic polymers Na polystyrene sulfonate Cholestyramine siliconeresin Carboxymethylcellulose Diethyl amino ethyl zein Poly aspartic acidcellulose Lignin Poly glutamic acid Poly citrulline fatty acids Polyornithine phospholipids gelatin divalent cations (e.g., calcium,magnesium, zinc)

[0110] In certain particularly preferred embodiments of this aspect ofthe invention, the microspheres include: (1) a carrier protein; (2) awater soluble polymer; (3) a first complexing agent that is apolyanionic polysaccharide such as dextran sulfate, galacturonic acids,alginates, mannuronic acid, guluronic acid, hyaluronic acid, chondroitinsulfates, heparin, chitin, chitosan, glycosaminoglycans, proteoglycans)and cationic complexing agents (i.e., complexing agents having apositive charge); and (4) a second complexing agent that is a divalentmetal cation selected from the group consisting of calcium, magnesium,zinc, strontium, barium, manganese, and iron. In the preferredembodiments of this aspect, the carrier protein is an albumin or animmunoglobulin or other protein selected from the group consisting ofthe carrier proteins of Table 1. In general, the microspheres of thisaspect of the invention contain from about 40 to less than 100% protein.In the preferred embodiments of this aspect, the water soluble polymeris selected from the group consisting of the water soluble polymers ofTable 2, preferably a carbohydrate-based polymer, and more preferably, ahydroxyethylstarch.

[0111] As with other aspects of the invention, these microspherespreferably have a smooth surface which includes a plurality of channelopenings that are less than 1000 angstroms as determined by gasadsorption technique for pore sizing and, preferably, do not containdetectable oil or organic solvent.

[0112] A preferred method of incorporating an ionic complexing agent(s)is to combine the ionic complexing(s) with a water soluble polymer inaqueous solution and the protein in aqueous solution and stabilize themicrosphere with heat or with crosslinking agents.

[0113] In general, the complexing agent is an ionic complexing agent(i.e., the complexing agent is capable of an ionic interaction or anon-ionic complexing agent (i.e., the complexing agent is capable of anon-ionic (e.g., hydrophobic) interaction or an agent with both ionicand nonionic interactions (e.g., IgG). Exemplary non-ionic complexingagents and ionic complexing agents such as anionic complexing agents(i.e., complexing agents having a negative charge) and cationiccomplexing agents (i.e., complexing agents having a positive charge) areprovided in Table 3.

[0114] In certain embodiments, the complexing agent is an anioniccomplexing agent having the structure of formula I:

POLY-[Y⁻]_(n)X⁺  I.

[0115] wherein POLY represents a principal chain of the anioniccomplexing agent which may be linear or branched;

[0116] wherein Y⁻ represents an anionic group, e.g., sulfates,carboxyls, phosphates, nitrates, carbonates and the like, that may becoupled to any one or more of the branches of the principal chain;

[0117] wherein X⁺ represents a cationic group, e.g., that is a counterion to the anionic group;

[0118] wherein n is an integer from 1 to 10,000, preferably, from 5 to100 and, more preferably, from 5 to 1000, and still more preferably,from 5 to 10,000; and

[0119] wherein when n is greater than 1, the n Y⁻ groups can be the sameor different.

[0120] In yet other embodiments of the invention the complexing agent isa cationic complexing agent having the structure of formula II:

POLY-[X⁺]_(n)Y⁻  II.

[0121] wherein POLY represents a principal chain of the cationiccomplexing agent which may be linear or branched;

[0122] wherein X⁺ represents a cationic group, e.g., an amino group,that may be coupled to any one or more of the branches of the principalchain;

[0123] wherein Y⁻ represents an anionic group, that is a counter ion tothe cationic group;

[0124] wherein n is an integer from 1 to 10000, preferably, from 5 to100 and, more preferably, from 5 to 1000, and most preferably, from 5 to10,000; and

[0125] wherein when n is greater than 1, the n X⁻ groups can be the sameor different.

[0126] Active Agents

[0127] According to another aspect of the invention, a microspherefurther including an active agent is provided. Exemplary categories ofactive agents are provided in Table 4. TABLE 4 Therapeutic Agents:Categories THERAPEUTIC AGENTS - CATEGORIES hormones, antibiotics andother antiinfective agents, hematopoietics, thrombopoictics, agents,antidementia agents, antiviral agents, antitumoral agents(chemotherapeutic agents), antipyretics, analgesics, anti- inflammatoryagents, antiulcer agents, antiallergic agents, antidepressants,psychotropic agents, cardiotonics, antiarrythmic agents, vasodilators,antihypertensive agents such as hypotensive diuretics, antidiabeticagents, anticoagulants, cholesterol lowering agents, therapeutic agentsfor osteoporosis, enzymes, vaccines, immunological agents and adjuvants,cytokines, growth factors, nucleotides and nucleic acids; carbohydratesand polysaccharides; viruses and virus particles; conjugates orcomplexes of small molecules and proteins, or mixtures thereof; andorganic or inorganic synthetic pharmaceutical drugs.

[0128] The microspheres into which the active agent can be loaded mayinclude a complexing agent to facilitate loading and/or modify therelease of the active agent from the microsphere. Alternatively, theactive agent can be loaded into the above-described microspheres whichlack a complexing agent, e.g., the protein and/or the water solublepolymers of the invention can interact with the active agent tofacilitate loading and/or modify its release from the microsphere. Ingeneral, although the active agent can be loaded into a microsphere ofthe invention during preparation of the microsphere, it is preferable toload the active agent into a preformed microsphere of the invention and,more preferable to load the active agent into a preformed microspherewhich contains a complexing agent(s) to facilitate loading and/orsustained release of the agent. In contrast to hydrogel microspheres,the microspheres of the invention do not swell significantly in waterand, further, the stabilized microspheres do not require swelling inorder to provide sustained release of the therapeutic protein and/orphysiologically active agent from the microsphere.

[0129] As used herein, an active agent refers to an agent which has adiagnostic or therapeutic activity. Accordingly, an active agentoptionally includes a detectable label (e.g., a radioactive label) thatis useful for identifying the locations of the released agent in vivo;Active agents also include therapeutic agents which are useful fortreating a disease or condition. In certain embodiments, the preferredphysiologically active agents are protein or peptide agents. Suchprotein or peptide agents typically can be further divided intocategories, based upon the activity of the agent or the type of diseaseor condition that is being treated. The physiologically active agentwhich can be used in the present invention includes but is not limitedto categories of antibiotics, hematopoietic agents, antiinfectiveagents, antidementia agents, antiviral agents, antitumoral agents,antipyretics, analgesics, antiinflammatory agents, antiulcer agents,antiallergic agents, antidepressants, psychotropic agents, cardiotonics,antiarrythmic agents, vasodilators, antihypertensive agents such ashypotensive diuretics, antidiabetic agents, anticoagulants, cholesterollowering agents, therapeutic agents for osteoporosis, hormones, vaccinesand so on (see, e.g., Table 4). While specific examples of active agents(e.g., peptide and non-peptide agents) for use in accordance with thisinvention are mentioned below, this does not mean that other peptide ornon-peptide agents are excluded. These active agents may be naturallyoccurring, recombinant or chemically synthesized substances.

[0130] The physiologically active agents of the invention includeprotein or peptide agents, as well as non-protein or non-peptide agents.For ease of discussion, such protein or peptide agents are referred tocollectively herein as peptide agents; non-protein and non-peptideagents shall be referred to collectively herein as non-peptide agents.Exemplary non-peptide agents include the following non-limitingcategories of agents: a) nucleotides and nucleic acids; b) carbohydratesand polysaccharides; c) viruses and virus particles; d) conjugates orcomplexes of small molecules and proteins, or mixtures thereof; and e)organic or inorganic natural or synthetic pharmaceutical drugs. Afurther description of these and other agents that can be used inaccordance with the methods and compositions of the present inventionare described in U.S. Pat. Nos. 5,482,706; 5,514,670; and 4,357,259, theentire contents of which are incorporated herein by reference.

[0131] The preferred physiologically active peptide agents includepeptide hormones, cytokines, growth factors, factors acting on thecardiovascular system, factors acting on the central and peripheralnervous systems, factors acting on humoral electrolytes and hemalorganic substances, factors acting on bone and skeleton, factors actingon the gastrointestinal system, factors acting on the immune system,factors acting on the respiratory system, factors acting on the genitalorgans, and enzymes.

[0132] Exemplary hormones include insulin, growth hormone, parathyroidhormone, luteinizing hormone-releasing hormone (LH-RH),adrenocorticotropic hormone (ACTH), amylin, oxytocin, luteinizinghormone, (D-Tryp6)-LHRH, nafarelin acetate, leuprolide acetate, folliclestimulating hormone, glucagon, prostaglandins, PGE1, PGE2 and otherfactors acting on the genital organs and their derivatives, analogs andcongeners. As analogs of said LH-RH, such known substances as thosedescribed in U.S. Pat. Nos. 4,008,209, 4,086,219, 4,124,577, 4,317,815and 5,110,904 can be mentioned.

[0133] Exemplary antibiotics include tetracycline, aminoglycosides,penicillins, cephalosporins, sulfonamide drugs, chloramphenicol sodiumsuccinate, erythromycin, vancomycin, lincomycin, clindamycin, nystatin,amphotericin B, amantidine, idoxuridine, p-amino salicyclic acid,isoniazid, rifampin, antinomycin D, mithramycin, daunomycin, adriamycin,bleomycin, vinblastine, vincristine, procarbazine, imidazolecarboxamide.

[0134] Exemplary hematopoictic or thrombopoictic factors include, amongothers, erythropoietin, granulocyte colony stimulating factor (G-CSF),granulocyte-macrophage stimulating factor (GM-CSF) and macrophage colonystimulating factor (M-CSF), leukocyte proliferation factor preparation(Leucoprol, Morinaga Milk), thrombopoietin, platelet proliferationstimulating factor, megakaryocyte proliferation (stimulating) factor,and factor VIII.

[0135] Exemplary Antidementia Agents Include Selegelene.

[0136] Exemplary antiviral agents include amantidine and proteaseinhibitors.

[0137] Exemplary antitumoral agents include doxorubicin, Daunorubicin,taxol, and methotrexate. Exemplary antipyretics and analgesics includeaspirin, Motrin, Ibuprofin, naprosyn, Indocin, and acetaminophen.

[0138] Exemplary antiinflammatory agents include NSAIDS, aspirin,steroids, dexamethasone, hydrocortisone, prednisolone, and DiclofenacNa.

[0139] Exemplary antiulcer agents include famotidine, cimetidine,nizatidine, ranitidine, and sucralfate.

[0140] Exemplary antiallergic agents include antihistamines,diphenydramine, loratadine, and chlorpheniramine.

[0141] Exemplary antidepressants and psychotropic agents includelithium, amitryptaline, tricyclic antidepressants, fluoxetine, prozac,and paroxetine.

[0142] Exemplary cardiotonics include digoxin.

[0143] Exemplary antiarrythmic agents include metoprolol andprocainamide.

[0144] Exemplary vasodilators include nitroglycerin, nifedipine, andIsosorbide dinitrate.

[0145] Exemplary diuretics include hydrochlorothiazide and furosemide.

[0146] Exemplary antihypertensive agents include captopril, nifedipine,and atenolol.

[0147] Exemplary antidiabetic agents include glucozide, chloropropamide,metformin, and insulin.

[0148] Exemplary anticoagulants include warfarin, heparin, and Hirudin.

[0149] Exemplary cholesterol lowering agents include lovastatin,cholestyamine, and clofibrate.

[0150] Exemplary therapeutic agents for treating osteoporosis and otherfactors acting on bone and skeleton include calcium, alendronate, boneGLa peptide, parathyroid hormone and its active fragments (osteostatin,Endocrinology 129, 324, 1991), histone H4-related bone formation andproliferation peptide (OGP, The EMBO Journal 11, 1867, 1992) and theirmuteins, derivatives and analogs thereof.

[0151] Exemplary enzymes and enzyme cofactors include: pancrease,L-asparaginase, hyaluronidase, chymotrypsin, trypsin, tPA,streptokinase, urokinase, pancreatin, collagenase, trypsinogen,chymotrypsinogen, plasminogen, streptokinase, adenyl cyclase, andsuperoxide dismutase (SOD).

[0152] Exemplary vaccines include Hepatitis B, MMR (measles, mumps, andrubella), and Polio vaccines.

[0153] Exemplary immunological adjuvants include: Freunds adjuvant,muramyl dipeptides, concanavalin A, BCG, and levamisole.

[0154] Exemplary cytokines include lymphokines, monokines, hematopoieticfactors and so on. Lymphokines and cytokines useful in the practice ofthe invention include interferons (e.g., interferon-alpha, -beta and-gamma), interleukins (e.g. interleukin 2 through 11) and so on.Monokines useful in the practice of the invention include interleukin-1,tumor necrosis factors (e.g. TNF-alpha and -beta), malignant leukocyteinhibitory factor (LIF) and so on.

[0155] Exemplary growth factors include nerve growth factors (NGF,NGF-2/NT-3), epidermal growth factor (EGF), fibroblast growth factor(FGF), insulin-like growth factor (IGF), transforming growth factor(TGF), platelet-derived cell growth factor (PDGF), hepatocyte growthfactor (HGF) and so on.

[0156] Exemplary factors acting on the cardiovascular system includefactors which control blood pressure, arteriosclerosis, etc., such asendothelins, endothelin inhibitors, endothelin antagonists described inEP 436189, 457195, 496452 and 528312, JP [Laid Open] No. H-3-94692/1991and 130299/1991, endothelin producing enzyme inhibitors vasopressin,renin, angiotensin I, angiotensin II, angiotensin III, angiotensin Iinhibitor, angiotensin II receptor antagonist, atrial naturiureticpeptide (ANP), antiarrythmic peptide and so on.

[0157] Exemplary factors acting on the central and peripheral nervoussystems include opioid peptides (e.g. enkephalins, endorphins),neurotropic factor (NTF), calcitonin gene-related peptide (CGRP),thyroid hormone releasing hormone (TRH), salts and derivatives of TRH[JP [Laid Open] No. 50-121273/1975 (U.S. Pat. No. 3,959,247), JP [LaidOpen] No. 52-116465/1977 (U.S. Pat. No. 4,100,152)], neurotensin and soon.

[0158] Exemplary factors acting on the gastrointestinal system includesecretin and gastrin.

[0159] Exemplary factors acting on humoral electrolytes and hemalorganic substances include factors which control hemaglutination, plasmacholesterol level or metal ion concentrations, such as calcitonin,apoprotein E and hirudin. Laminin and intercellular adhesion molecule 1(ICAM 1) represent exemplary cell adhesion factors.

[0160] Exemplary factors acting on the kidney and urinary tract includesubstances which regulate the function of the kidney, such asbrain-derived naturiuretic peptide (BNP), urotensin and so on.

[0161] Exemplary factors which act on the sense organs include factorswhich control the sensitivity of the various organs, such as substanceP.

[0162] Exemplary factors acting on the immune system include factorswhich control inflammation and malignant neoplasms and factors whichattack infective microorganisms, such as chemotactic peptides andbradykinins.

[0163] Exemplary factors acting on the respiratory system includefactors associated with asthmatic responses.

[0164] Also included are naturally occurring, chemically synthesized orrecombinant peptides or proteins which may act as antigens, such ascedar pollen and ragweed pollen. These factors are administered, eitherindependently, coupled to haptens, or together with an adjuvant, in theformulations according to the present invention.

[0165] Microsphere Formation, Stabilization, and Characterization

[0166] The method for forming the microspheres of the inventioninvolves: (1) combining, in one or more aqueous solutions, amacromolecule such as a protein, a water soluble polymer (e.g.,carbohydrate-based polymer) and a complexing agent(s) to form an aqueousmixture (either as a single or multi-phase system); and, preferably, (2)subjecting the aqueous mixture to a crosslinking agent (e.g., EDC[1-ethyl-3-(3-dimethylamino-propylcarbodiimide]) and/or an energy sourcefor a time sufficient to form a microsphere, particularly, a microspherehaving a smooth surface which includes a plurality of channel openingsthat are less than 1000 angstroms in diameter as determined by gasadsorption technique for pore sizing. Exemplary methods of preparing themicrospheres are provided in the Examples.

[0167] The particularly preferred embodiments are microspheres whichinclude:

[0168] (1) a carrier protein;

[0169] (2) a water soluble polymer;

[0170] (3) a first complexing agent that is a poly anionicpolysaccharide; and

[0171] (4) a second complexing agent that is a divalent metal cation,preferably calcium or magnesium. Active agents, such as therapeuticagents or diagnostic agents, can be introduced into these microspheresfollowing their formation. The preferred method for forming theseparticularly preferred embodiments of the invention involves: (1)combining, essentially simultaneously, in one or more aqueous solutions,the carrier protein, the water soluble polymer, the first complexingagent, and the second complexing agent to form an aqueous mixture; and(2) allowing the microspheres to form. Optionally, the microspheres canbe further subjected to a cross-linking agent to further stabilize themicrospheres. By “essentially simultaneously,” it is meant that thesecond complexing agent (preferably calcium or magnesium) is addedwithin about 30 minutes of the addition of the other forming components.Applicants have discovered that the addition of calcium or magnesium tothe aqueous mixture which is not an essentially simultaneous additionresults in the formation of aggregates and other amorphous forms ofparticles, rather than the spherically shaped microspheres which areformed when the calcium or magnesium is combined essentiallysimultaneously with the other components during the microsphereformation process.

[0172] Microspheres can be stabilized by incubation of the formedmicrospheres in the presence of a crosslinking agent and/or an energysource (e.g., heat) for a predetermined length of time. Exemplarycrosslinking agents include dialdehydes, amines, multivalent ions,multifunctional molecules having an affinity for specific reactivegroups on the macromolecule being crosslinked, N-substituted maleimides,bifunctional alkyl halides, aryl halides, isocyanates, aliphatic oraromatic dicarboxylic acids, aliphatic or aromatic disulphonic acids,bifunctional imidoesters, and vinylsulphones. Additional crosslinkingagents and methods for using same to stabilize a microsphere aredescribed in U.S. Pat. No. 5,578,709, issued to J. Woiszwillo, theentire contents of which are incorporated in their entirety herein byreference. The preferred energy source is heat. However, it will beunderstood by those skilled in the art that other energy sources includeheat, radiation, and ionization, alone or in combination withsonication, vortexing, mixing or stirring. Microsphere formation and/orstabilization can occur immediately upon exposure to the energy sourceor may require an extended exposure to the energy source depending onthe characteristics of the components and conditions. Preferably, themacromolecule-polymer solution mixture, is incubated in a water bath ata temperature greater than or equal to 37° C. and less than or equal to90° C. for between approximately 5 minutes and 2 hours. Most preferably,the mixture is incubated for 5-30 minutes at a temperature between 50°C. and 90° C. It should be noted that microspheres may be formed atlower temperatures by utilizing a higher macromolecule concentration.The maximum incubation temperature is determined by the characteristicsof the macromolecule and the ultimate function of the microsphere. Forexample, for a microsphere in which the macromolecule is a protein, atemperature less than approximately 70° C. is preferred to retainprotein activity.

[0173] The formed microspheres are separated from the non-incorporatedcomponents of the incubation mixture by conventional separation methodswell known to those skilled in the art. Preferably, the incubationmixture is centrifuged so that the microspheres sediment to the bottomof the centrifuge tube and the non-incorporated components remain in thesupernatant, which is then removed by decanting. Alternatively, asuspension containing formed microspheres is filtered so that themicrospheres are retained on the filter and the non-incorporatedcomponents pass through the filter.

[0174] Further purification of the microspheres is achieved by washingin an appropriate volume of a washing solution. The preferred washingsolution is a buffer, most preferably a nonionic aqueous solution or anonionic aqueous solution containing water soluble polymers. Repeatedwashings can be utilized as necessary and the microspheres separatedfrom the wash solution as described above.

[0175] As mentioned above, the characteristics of the microspheres canbe altered by manipulating the incubation conditions. For example, therelease kinetics of the microspheres may be retarded by increasing thereaction temperature or extending the length of reaction time duringmicrosphere formation. Release kinetics are also manipulated by choosingdifferent polymers, different concentrations of polymers, or differentratios of polymers used in the formation of the microspheres.

[0176] Microsphere size, shape and release kinetics can also becontrolled by adjusting the microsphere formation conditions. Forexample, particle formation conditions can be optimized to producesmaller or larger particles or the overall incubation time or incubationtemperature can be increased, resulting in particles which haveprolonged release kinetics.

[0177] Nucleic Acid Microspheres

[0178] According to still other embodiments of the invention,microspheres in which the macromolecule is a nucleic acid are provided.The nucleic acid-containing microspheres include: (1) a nucleic acid(e.g., plasmid, viral vector, oligonucleotide, RNA, antisense andmissense nucleic acids); (2) a polycationic polymer(s) (e.g.,polylysine); and (3) a water soluble polymer (as described above). Thus,according to a related aspect of the invention, a method for forming thenucleic acid-containing microspheres is provided. The method involves:(1) combining, in one or more aqueous solutions, a nucleic acid(s), apolycationic polymer(s) and a water soluble polymer(s) to form anaqueous mixture; and (2) subjecting the aqueous mixture to acrosslinking agent and/or an energy source for a time sufficient to forma microsphere. Exemplary methods of forming the nucleic acidmicrospheres are provided in the Examples.

[0179] Pharmaceutical Compositions

[0180] According to yet another aspect of the invention, apharmaceutical composition of matter and method for producing same areprovided. The composition includes a container containing a single doseof microspheres containing an active agent for treating a condition thatis treatable by the sustained release of an active agent from themicrospheres. The number of microspheres in the single dose is dependentupon the amount of active agent present in each microsphere and theperiod of time over which sustained release is desired. Preferably, thesingle dose is selected to achieve the sustained release of the activeagent over a period of about 1 to about 180 days with the desiredrelease profile.

[0181] According to another aspect of the invention, asyringe-containing composition is provided. The composition includes asyringe containing a single dose of microspheres containing an activeagent for treating a condition that is treatable by the sustainedrelease of the active agent form the microspheres; and a needle attachedto the syringe, wherein the needle has a bore size that is from 14 to 30gauge.

[0182] Remarkably, the preferred microspheres of the invention can beprepared to have a dimension which permits the delivery of microspheresusing a needleless syringe (MediJector, Derata Corporation, Minneapolis,Minn. 55427), thereby eliminating the disposal problems inherent toneedles which must be disposed as a biohazard waste product. Thus,according to a particularly preferred aspect of the invention, aneedleless syringe containing one or more doses of microspherescontaining an active agent for treating a condition is provided. Themicrospheres can be prepared to have qualities suitable to be deliveredby other parenteral and non-parenteral routes such as oral, buccal,intrathecal, nasal, pulmonary, transdermal, transmucosal and the like.

[0183] Utilities

[0184] In summary, the compositions of the invention can be thought ofas combining various components, as exemplified in the Tables, underconditions to form microspheres which, preferably, having desirablesmooth surface characteristics that are quantifyable in terms ofdetermining whether the microspheres have formed (e.g., by visualdetection) and in terms of determining the diameter of the microspherechannel openings. Thus, in certain of its broadest aspect, thecompositions of the invention are directed to microspheres having therequisite channel opening dimensions and comprising a macromolecule(preferably a protein of Table 1) and a water soluble polymer(preferably a polymer of Table 2). In yet another aspect, themicrospheres comprise a macromolecule (preferably a protein of Table 1),a water soluble polymer (preferably a polymer of Table 2), and acomplexing agent (preferably of Table 3). In the particularly preferredembodiments, the microspheres include: (1) a carrier protein; (2) awater soluble polymer; (3) a first complexing agent that is apolyanionic polysaccharide; and (4) a second complexing agent that is adivalent metal cation selected from the group consisting of calcium andmagnesium. In still further aspects, the invention is directed to any ofthese aspects wherein the microspheres further comprise a therapeuticagent (preferably of Table 4). Thus, in certain embodiments of theseaspects, a protein(s) of Table 1 is combined with a water solublepolymer(s) of Table 2 and an active agent(s) of Table 4. In yet otherembodiments of these aspects, a nucleic acid(s) is combined with apolycation(s) of Table 3, a water soluble polymer(s) of Table 2 and,optionally, a therapeutic agent(s) of Table 4. Accordingly, the methodsof the invention provide various microspheres which can be designed fordifferent therapeutic and diagnostic applications by selecting theappropriate combination of agents for achieving the therapeutic ordiagnostic goal.

[0185] When used therapeutically, the microspheres of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means that amount of active agentnecessary to delay the onset of, inhibit the progression of, or haltaltogether the particular condition being treated. Generally, atherapeutically effective amount will vary with the subject's age,condition, and sex, as well as the nature and extent of the disease inthe subject, all of which can be determined by one of ordinary skill inthe art. The dosage may be adjusted by the individual physician orveterinarian, particularly in the event of any complication. Atherapeutically effective amount of active agent typically varies from0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about200 mg/kg, and most preferably from about 0.2 mg//kg to about 20 mg/kg,in one or more dose administrations daily, for one or more days, weekly,monthly, every two or three months, and so forth.

[0186] The microspheres may be administered alone or in combination withother drug therapies as part of a pharmaceutical composition. Such apharmaceutical composition may include the microspheres in combinationwith any standard physiologically and/or pharmaceutically acceptablecarriers which are known in the art. The compositions should be sterileand contain a therapeutically effective amount of the microsphere in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically-acceptable carrier” as used herein means one ormore compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration into a human or otheranimal. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficacy. Pharmaceutically acceptable further means anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, desiccants, bulking agents, propellants,acidifying agents, coating agents, solubilizers, and other materialswhich are well known in the art. Carrier formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. administrations can befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

[0187] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular drug selected,the severity of the condition being treated, and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Oraladministration will be preferred for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule.

[0188] The pharmaceutical compositions may conveniently be presented inunit dosage form and may be prepared by any of the methods well-known inthe art of pharmacy. All methods include the step of bringing themicrosphere into association with a carrier which constitutes one ormore accessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the microspheres into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

[0189] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Additionalexamples of solvents include propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, salts and buffer solutionssuch as saline and buffered media, alcoholic/aqueous solutions andemulsions or suspensions. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. In general, the microspheres canbe administered to the subject (any mammalian recipient) using the samemodes of administration that currently are used for microparticletherapy in humans. The microspheres are useful for a wide variety ofseparations, diagnostic, therapeutic, industrial, commercial, cosmetic,and research purposes as discussed in more detail below. For example,for in vivo diagnostic purposes, the microspheres can include amacromolecule such as an immunoglobulin or cell receptor labeled with adetectable label. Administration of the labeled microsphere to a patientcreates an imaging agent for the diagnosis of a proliferative disordersuch as cancer or a tool for the evaluation of the success of atherapeutic agent in reducing the proliferation of a particular adversecell or organism.

[0190] For in vitro diagnosis, microspheres containing a macromolecule,such as an immunoglobulin, cell receptor or oligonucleotide probespecific for the cell or organism under investigation, are combined witha test sample, the microspheres are separated from any non-boundcomponents of the sample, and bound molecules are detected byconventional methods.

[0191] The microspheres are useful as therapeutic agents and may enablethe use of alternative routes of administration when the microspheresinclude a therapeutic drug and are administered to a patient for slowrelease or targeted delivery of the drug to the site requiring therapy.The microspheres are also useful as therapeutic or prophylactic agentswhen the microspheres include a macromolecule that is itself atherapeutic or prophylactic agent, such as an enzyme or immunoglobulin.The slow release of such therapeutic agents is particularly useful fortherapeutic proteins or peptides having short half-lives that must beadministered by injection.

[0192] The microspheres are also useful for the purification ofmolecules from a complex mixture, as a reagent for the detection orquantification of a specific molecule, or for the production ofmolecules, such as antibodies. For example, microspheres containing amacromolecule, such as an immunoglobulin, can be attached to achromatography column and used in immunoaffinity chromatography toseparate a ligand from a complex mixture. Alternatively, microspheresincluding a labeled macromolecule or a mixture of labeled macromoleculesspecific for different cells or biomolecules, such as cell receptors,can be used to detect changes in the number of cells or biomolecules inresponse to a particular test condition using techniques such as flowcytometry.

[0193] Furthermore, the microspheres can be used as adjuvants forvaccine production wherein antigen-containing microspheres are injectedinto a research animal, such as a mouse or rabbit, to trigger anenhanced immune response for the production of antibodies to theantigen.

[0194] Additional commercial uses include cleaning formulations, such asthe formation of enzyme particles for addition to detergents; cosmetics,such as the formation of collagen particles to be suspended in a lotionor cream; ink; and paint.

[0195] In Vitro Diagnostics

[0196] In Vitro Assays: The microspheres described herein are useful assolid phase particles in an assay, such as an enzyme-linkedimmunosorbant assay, dot-blot, or Western blot, for the detection of aparticular target such as a cell, biomolecule or drug in a biologicalsample. The microspheres designed for this use are composed of affinitymolecules specific for the target molecule. For example, themacromolecule is an immunoglobulin, cell receptor or oligonucleotideprobe and is bound to a test tube or microtiter plate.

[0197] For detection or quantitation of a target molecule of interest, asample is combined with a solution containing the microspheres, themacromolecules on the microspheres are reacted with the target molecule,the microspheres are separated from any non-bound components of thesample, and microspheres containing bound molecules are detected byconventional methods. Fluorescently stained microspheres areparticularly well suited for flow cytometry analysis in accordance withmethods well known to those skilled in the art.

[0198] Histopathology: The microspheres described herein are useful asvisual probes or markers of pathology in a histological sample. Themacromolecules of microspheres designed for this use are specific forbiomolecules expressed during a particular pathologic condition and arelabeled with a detectable label. For example, the macromolecule is animmunoglobulin, cell receptor or oligonucleotide probe specific for anabnormal cell, such as a rapidly proliferating cell, or pathologicalorganism, for example, a virus.

[0199] For detection of a pathogenic condition, a histological sample iscombined with a solution containing the microspheres, the labeledmacromolecules on the microspheres are reacted with the target moleculeof interest, and bound microspheres are detected by detecting the labelin accordance with methods well known to those skilled in the art.

[0200] In Vivo Diagnostics-Imaging

[0201] The microspheres described herein are useful as imaging agentsfor in vivo localization of a particular molecule, cell type orpathologic condition in a manner similar to that described above withregard to the use of the microspheres for histopathology. Themacromolecules on microspheres designed for this use are specific formolecules expressed by a particular cell or pathologic organism and arelabeled with a detectable label. For example, the macromolecule is animmunoglobulin, cell receptor or oligonucleotide probe specific for atumor cell or pathological organism, such as a virus.

[0202] The microspheres are used to either detect a pathologic conditionor to monitor the success of therapy, such as chemotherapy or surgery toensure that the size of an abnormal tissue tumor has decreased or hasbeen completely excised. For this use, a patient receives anadministration of a microsphere solution, preferably intravenously, thelabeled macromolecules on the microspheres are given a sufficient amountof time to localize to the affected organ or region of the body, themacromolecule is reacted with a target molecule expressed by the cell ororganism under investigation, and bound microspheres are detected bydetecting the label by conventional imaging techniques well known tothose skilled in the art, such as x-ray.

[0203] Drug Delivery Systems

[0204] The microspheres are useful for therapy or prophylaxis when themacromolecule is a therapeutic agent or a pharmaceutical compound thatis delivered to a patient and slowly released from the microspheres overtime. These microspheres are particularly useful for slow release ofdrugs with short biological half-lives, such as proteins or peptides. Ifthe pharmaceutical compound cannot be formed into a particle, then it iscomplexed to a carrier, such as albumin, and the carrier-pharmaceuticalcompound complex is formed into a microsphere. The microsphere caneither provide for the slow release of the agent throughout the body orthe microsphere can include an affinity molecule specific for a targettissue, or tumor, and be injected into a patient for targeted slowrelease of the therapeutic agent, such as an antitumor, antiviral,antibacterial, antiparasitic, or aintiarthritic agent, cytokine,hormone, or insulin directly to the site requiring therapy. As discussedabove, the affinity molecule may be cleavable.

[0205] Microspheres composed of antigenic proteins orpolysaccharide-protein conjugates capable of provoking an immuneresponse are particularly suitable for use as vaccines.

[0206] The microspheres are also useful as vehicles for gene therapy orthe production of “genetic vaccines” when composed of nucleic acids,such as DNA or RNA, that are either incorporated into the DNA of thepatient or are transfected into a target cell to produce a desiredprotein. For example, polynucleotides encoding core proteins of virusessuch as influenza or human immunodeficiency virus HIV can be deliveredas microspheres for expression of an antigenic protein. This isadvantageous in that new vaccines need not be developed as often becauseviral core proteins mutate to a much lesser extent than the cell surfaceantigens currently used in vaccines. The nucleic acid microspheres aredelivered to mammalian cells in much the same way as naked DNA isdelivered. The desired nucleic acid sequence is inserted into a vector,such as plasmid DNA, with a promoter, such as the SV40 promoter or thecytomegalovirus promoter, and optionally may include a reporter gene,such as beta-galactosidase. The nucleic acid is preferably combined witha carrier protein and/or a cation, such as polylysine, to facilitateparticle formation as described above. The microspheres are thenadministered directly to the patient or are transfected into mammaliancells that are then administered to the patient requiring therapy orprophylaxis. The nucleic acid microspheres may include a substance suchas chloroquine, which allows nucleic acids to escape from cytoplasmiccompartments into the cytoplasm so that it can be more easilytranscribed and translated by the cells. Additionally, the microspheresmay be coated with a substance that increases the efficiency oftranslation or may be coated with a substance to provide cell-specifictargeting of the microspheres.

[0207] Research Applications

[0208] The microspheres are useful as research tools for thepurification of a biomolecule from a complex mixture, as a reagent forthe detection or quantification of a biomolecule, or for the productionof biomolecules, such as antibodies.

[0209] For example, microspheres composed of a macromolecule, such as animmunoglobulin, are attached to a chromatography column and used inimmunoaffinity chromatography to separate a ligand from a complexmixture. It will be understood by those skilled in the art thatmicrosphere for use in high pressure liquid chromatography should befirst attached to a non-compressible solid phase sphere or bead so thatthe column packing maintains its rigid structure under pressure.

[0210] Alternatively, microspheres including a labeled macromolecule ora mixture of labeled macromolecules specific for different cells or cellreceptors are used to detect changes in the number of cells or cellsurface receptors in response to a particular test condition usingtechniques such as flow cytometry.

[0211] The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Preparation of Microspheres Containing aPharmaceutical Agent

[0212] Human serum albumin (HSA) Microspheres suitable for use as a drugdelivery vehicle were prepared.

[0213] Preparation of Rifampicin TM-HSA Microspheres

[0214] Carbonyl diimidazole (124 mg, Sigma Chemical Co., St. Louis, Mo.)was added to a solution of 50 mg of the antibiotic Rifampicin TM(3-[4-methylpiperazinyl-iminomethyl] rifamycin, Sigma Chemical Co., St.Louis, Mo.) in 2 ml dimethylformamide (DMF). The resulting mixture wasallowed to stand at room temperature for four hours. To the mixture wasadded a mixture of 1 ml of human serum albumin (HSA, 25%, ArmourPharmaceutical Co., Collegeville, Pa.) and 2 ml deionized water. Themixture was left at room temperature overnight. 14 ml of a polymersolution containing 25% PVP (40,000 daltons) and 25% PEG (3,350 daltons)in 0.1M NaOAc, pH 4 was added to the mixture. The mixture was incubatedfor 30 minutes at room temperature, for 30 minutes at 37° C., and for 30minutes at 58° C. and then cooled to room temperature. Particles wereisolated by centrifugation, washed with deionized water three times andresuspended in 20 ml of water. The percentage of HSA incorporated intothe particles was 74% (assayed by the BCA TM protein assay (Pierce,Rockford, Ill.)). The percentage of Rifampicin TM incorporated into theparticles was greater than 6.8%. The average size of the particles wasdetermined to be 68 nm in diameter using a Coulter TM cell sorter.

[0215] Preparation of Virazole TM-HSA Microspheres

[0216] Carbonyl diimidazole (100 mg, Sigma Chemical Co., St. Louis, Mo.)was added to a solution of 36 mg of the antiviral drug VirazoleRegistered TM (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.) in 0.2 mlof dimethylformamide (DMF). The resulting mixture was allowed to standat room temperature for four hours. To the mixture was added a mixtureof 0.2 ml of human serum albumin (HSA) (25%, Armour Pharmaceutical Co.,Collegeville, Pa.) and 0.4 ml of deionized water. The mixture wasincubated for 30 minutes at room temperature, for 30 minutes at 37° C.,and for 30 minutes at 58° C. and then cooled to room temperature.Particles were isolated by centrifugation, washed with deionized waterthree times and resuspended in 20 ml of water. The percentage of HSAincorporated into the particles was 61% (assayed by the BCA TM proteinassay (Pierce, Rockford, Ill.)). The percentage of Virazole RegisteredTM incorporated into the particles was 10%.

Example 2 Attachment of Polysaccharide to Outer Surface of ProteinMicrospheres

[0217] The polysaccharide PRP-AH was coupled to the outer surface of twodifferent protein microspheres.

[0218] An adipic acid dihydrazide derivative (AH) of thepolyribosylribitol phosphate (PRP) of Haemophilus influenza type b(Hib), one of the major causative organisms of bacterial meningitis,referred to as PRP-AH, was prepared by coupling PRP to adipic acid(Sigma Chemical Co., St. Louis, Mo.) in the presence of cyanogen bromide(Sigma Chemical Co., St. Louis, Mo.) (The PRP was obtained from theMassachusetts Public Health Biologic Laboratory (Jamaica Plain, Mass.)).

[0219] Coupling of PRP-AH to Ovalbumin Microspheres

[0220] Ovalbumin microspheres were prepared by adding ovalbumin (1%,Sigma Chemical Co., St. Louis, Mo.) to a polymer solution containing 25%PVP (40,000 daltons) and 25% PEG (3,500 daltons). The mixture wasincubated for 30 minutes at room temperature, 30 minutes at 37°, and 30minutes at 58° C., causing the formation of microspheres. The particleswere collected by centrifugation. The average diameter of the particleswas determined to be approximately 0.068 μm. The ovalbumin particles(1.5 mg) in 1M MES buffer, pH 5.0 (0.1 ml) were combined with the PRP-AH(1.5 mg). Subsequently, 2.79 mg of1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC, SigmaChemical Company, St. Louis, Mo.) were added. The reaction was mixed atroom temperature for three hours. The particles were collected bycentrifugation and washed three times with 1M MES buffer pH 5.0 (400 mul). The yield of PRP was determined to be 25% by the Anthrone freepolysaccharide assay described in METHODS IN IMMUNOLOGY ANDIMMUNOCHEMISTRY, Vol. II, Williams, C. A. and Chase, M. W.(eds.), 1968,pp. 288-289, Academic Press, New York. Protein content was determined tobe 55% by the BCA TM protein assay (Pierce, Rockford, Ill.). The ratioof PRP to protein was 0.46. The average diameter of the resultingparticles was 0.067 um.

[0221] The recovery of free polysaccharide and the loading ofpolysaccharide on the particles (polysaccharide:ovalbumin ratio) wasdependent on the starting ratio of polysaccharide to ovalbumin particle.As the ratio of polysaccharide to ovalbumin particle was increased, therecovery of free polysaccharide was decreased and the loading ofpolysaccharide onto the particles was increased as shown in ExampleTable 1 below.

Example

[0222] TABLE 1 Polysaccharide Recovery and Loading on OvalbuminMicrospheres Starting Ratio of Polysaccharide Polysaccharide: RecoveryLoading of Particle (%) Polysaccharide 1:1 25 0.46 1:2 30 0.29 1:4 660.21 1:8 94 0.14

[0223] Coupling of PRP-AH to Tetanus Toxoid Microspheres

[0224] Tetanus toxoid, (27 mg/ml, obtained from the Massachusetts PublicHealth Biologic Laboratory (Jamaica Plain, Mass.)) was combined with twovolumes of a polymer solution containing 25% PVP (40,000 daltons) and25% PEG (3,500 daltons), pH 5.0. The mixture was incubated for 30minutes at room temperature, 30 minutes at 37° C., and 30 minutes at 58°C., causing the formation of microspheres. The particles were collectedby centrifugation. The average diameter of the particles was determinedto be approximately 0.082 um.

[0225] The tetanus toxoid particles (0.825 mg) in 1M MES buffer, pH 5.0(0.1 ml) were combined with the PRP-AH (1.5 mg). Subsequently, 2.79 mgof 1-ethyl-3-(3-dimethyl-aminopropyl-carbodiimide hydrochloride (EDC,Sigma Chemical Company, St. Louis, Mo.) were added. The reaction wasmixed at room temperature for three hours. The particles were collectedby centrifugation and washed three times with 1M MES buffer pH 5.0 (400mu l). The yield of PRP was determined to be 16% by the Anthrone freepolysaccharide assay described in METHODS IN IMMUNOLOGY ANDIMMUNOCHEMISTRY, Vol. 11, Williams, C. A. and Chase, M. W.(eds.), 1968,pp. 288-289, Academic Press, New York. Protein content was determined tobe 99% by the BCA TM protein assay (Pierce, Rockford, Ill.). The ratioof PRP to protein was 0.1. The average diameter of the resultingparticles was 0.080 um.

Example 3 Release of Radiolabeled Protein and Polymer from Microspheres

[0226] Microspheres were prepared using Radiolabeled protein (bovineserum albumin, BSA) and Radiolabeled polymer (PEG). The release ofradioactivity was measured as a function of time.

[0227] Microspheres were prepared by combining Radiolabeled protein (10mg/ml [14]C-BSA, NEN, Boston, Mass.) with two volumes of a polymersolution containing 25%PVP (40,000 daltons) and 25% [3] H-PEG (3,500daltons, NEN, Boston, Mass.), pH 5.0. The mixture was incubated for 30minutes at 37° C., 30 minutes at 58° C., and 30 minutes at 70° C.causing the formation of microspheres. The particles were collected bycentrifugation.

[0228] Protein and polymer were slowly released from the microspheres byadding 500 ml of phosphate buffered saline (pH 7.4) and incubating themixture at 37° C., while shaking mildly using a Nutator TM rotator. Atvarious time points, particles were precipitated by centrifugation at8,000 rpm for 10 minutes, the supernatant was removed with a pipette,and radioactivity was assayed by adding liquid scintillation fluid andcounting in a liquid scintillation counter. The particles were thenresuspended in 500 ml of phosphate buffered saline (pH 7.4) and replacedat 37° C. with gentle rotation until the next time point. The releasekinetics of Radiolabeled protein and polymer during incubation at 37° C.is shown in FIG. 1.

[0229] Microspheres were prepared and assayed for release as describedabove, however, three different concentrations of polymer were used, andthe microspheres were formed by incubation at 58° C. In the firstpreparation, 25% PEG and 25% PVP were used. In the second preparation,20% PEG and 20% PVP were used. In the third preparation, 12.5% PEG and12.5% PVP were used. The release kinetics of Radiolabeled protein isshown in FIG. 2. The release kinetics of Radiolabeled polymer is shownin FIG. 3.

[0230] Microspheres were once again prepared and assayed for release asdescribed above, however, three different concentrations of polymer wereused, and the microspheres were formed by incubation at 58° C., 70° C.,both 37° C. and 58° C., and both 37° C. and 70° C. The release kineticsof Radiolabeled protein is shown in FIG. 4. The release kinetics ofRadiolabeled polymer is shown in FIG. 5. Radiolabeled PEG release as afunction of polymer concentration is shown in FIG. 6.

Example 4 Formation of DNA-Containing Microspheres

[0231] DNA-containing microspheres were prepared and transfected intofibroblast cells. The microspheres were analyzed for transfectionefficiency and protein expression.

[0232] A 0.025 mL aliquot of a 1 mg/mL solution of a plasmid DNA (pCMVbeta Gal, Promega, Milwaukee, Wis.) was complexed with 0.025 mL of a 5.0mg/mL solution of poly-L-lysine having an average molecular weight rangeof from 1 kDa to 40 kDa (Sigma Chemical Co., St. Louis, Mo.).

[0233] To the plasmid DNA-poly-L-lysine complex was added, whilevortexing, 0.1 mL of a solution of 25% (weight/volume)polyvinylpyrrolidone (average molecular weight 40 kDa, Spectrum,Gardena, Calif.) and 25% (weight/volume) polyethylene glycol (averagemolecular weight 3.35 kDa, Spectrum, Gardena, Calif.) in 0.1M sodiumacetate, pH 5.5.

[0234] The mixture was incubated at 37° C. for 30 minutes and then at70° C. for 30 minutes. DNA-containing microspheres were formed. Themixture was centrifuged at 17,500×g for 10 minutes, the supernatantaspirated, and the particles washed three times with 0.3 mL of 10%glycerol (volume/volume) in deionized water. The microspheres wereresuspended in 0.050 mL deionized water. The DNA-containing microsphereswere applied to NIH3T3 fibroblast cells and incubated up to 24 hours toallow microsphere uptake by the cells. Uptake was terminated by washingthe cells three times with phosphate buffered saline(PBS/Ca[2+]+Mg[2+]-free) (GIBCO-BRL, Gaithersburg, Md.) and the additionof Dulbecco's Minimal Essential Media (DMEM, GIBCO-BRL, Gaithersburg,Md.).

[0235] The uptake and expression of the pCMV beta Gal DNA was assayedfor efficiency of transfection and amount of expressedbeta-galactosidase enzyme. The efficiency of transfection was determinedby fixation of the cells and color development with thebeta-galactosidase enzyme substrate X-Gal(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, GIBCO-BRL,Gaithersburg, Md.). The amount of expressed beta-galactosidase enzymewas determined by lysing the transfected cells and measuring totalenzyme activity with the beta-galactosidase enzyme substrate CPRG(chlorphenolred-beta-D-galactopyranoside, Boehringer Mannheim,Indianapolis, Ind.)

[0236] Results: The amount of expressed beta-galactosidase enzyme fromlysed cells that were transfected using either: 1) naked DNA (noaddition); 2) cationic liposomes plus DNA; or 3) DNA-containingmicrosphere, prepared as described above, is shown in FIG. 7.

Example 5 Formation of Leuprolide Acetate-Containing Microspheres

[0237] Microspheres containing leuprolide acetate peptide and humanserum albumin were prepared. Leuprolide acetate is a generic analog ofluteinizing hormone releasing hormone, which is a peptide used primarilyin the treatment of prostate cancer.

[0238] A 0.010 mL aliquot of a solution of 10-100 mg/mL leuprolideacetate in water (LHRH, TAP Pharmaceuticals, Deerfield, Ill.) was addedto 0.168 mL of a 2-10% (weight/volume) solution of dextran sulfate inwater (average molecular weight 500 kDa), and the solution wasthoroughly mixed. To the leuprolide/dextran solution was added a 0.856mL aliquot of a solution containing 25% (weight/volume) polyethyleneglycol, having an average molecular weight of 3.35 kDa (Spectrum,Gardena, Calif.), and 25% (weight/volume) polyvinylpyrrolidone, havingan average molecular weight of 40 kDa, in an aqueous solution of 0.1Msodium acetate, pH 5.5. The resulting solution was thoroughly mixed andallowed to stand for up to 30 minutes. A 0.25 mL aliquot of a 20%(weight/volume) solution of human serum albumin (Sigma Chemical Co., St.Louis, Mo.) in water was then added to the solution. The final solutionwas thoroughly mixed and placed in a water bath at between 70° C. and90° C. for a period of time between 30 minutes and 3 hours. Microsphereswere formed.

[0239] The microspheres were collected by centrifugation at 17.5K×g for10 minutes, washed in 0.5 mL of dH2O, and collected again bycentrifugation.

[0240] Sterile particles were prepared using the foregoing procedure andby sterile filtering all solutions prior to use and conduction all opentube manipulations in a laminar flow tissue culture hood.

[0241] In vitro release of leuprolide acetate was measured bycentrifugation of microspheres and resuspension in a phosphate bufferedsaline release medium. The release kinetics are shown in FIG. 8.

[0242] The microspheres were composed of approximately 10% leuprolideacetate, 50% human serum albumin, 20% dextran sulfate and 20%polyethylene glycol/polyvinylpyrrolidone.

[0243] Similar particles were prepared which also included zinc sulfateor caprylic acid, both of which retarded the release of protein andpeptide from the microspheres.

Example 6 Preparation of Bovine Serum Albumin Microspheres UsingPolyethylene Glycol and Poloxamer 407

[0244] Bovine serum albumin microspheres were prepared using a polymermixture of polyethylene glycol and poloxamer 407. 1.25 grams ofpolyethylene glycol (MW 3550, Sigma Chemical Co., St. Louis, Mo.) andpoloxamer 407 (BASF, Parsippany, N.J.) were dissolved in 100 ml of a0.1N sodium acetate buffer, pH 5.5 to make a 12.5% solution. A solutionof 10 mg/ml bovine serum albumin (BSA, Fraction V, Sigma Chemical Co.)was dissolved in dH2O. A 400 ml aliquot of the BSA solution was combinedwith 800 ml of the polymer solution. The mixture was vortexed. A clearsolution formed. The solution was heated to 70° C. for 30 minutes.Particle formation was observed by the presence of a milky whitesuspension.

[0245] The residual polymer solution was removed by centrifugation at12,500 rpm for 10 minutes and then decanting the solvent. Two washes andcentrifugation steps with 10% ethanol in water were performed to removeadditional residual polymer.

Example 7 Preparation of Bovine Serum Albumin Microspheres Using Dextran

[0246] Bovine serum albumin microspheres were prepared using dextran.12.5 grams of dextran (MW 500,000, Sigma Chemical Co., St. Louis, Mo.)were dissolved in 100 ml of a 0.1M sodium acetate buffer, pH 5.0 to makea 12.5% solution. Bovine serum albumin (BSA, Sigma Chemical Co.) wasdissolved in dH2O at a concentration of 10 mg/ml. A 400 ml aliquot ofthe BSA solution was place in a 1.5 ml microcentrifuge tube. An 800 mlaliquot of the dextran polymer solution was added to the BSA solution.The solution was vortexed. A clear solution formed. The microcentrifugetube was place in a 70° C. water bath for 30 minutes. A milky whitesuspension was observed, indicating microsphere formation.

[0247] Residual dextran polymer solution was removed by centrifugationat 12,500 rpm for 10 minutes and then decanting the solvent. Two washesand centrifugation steps with 10% ethanol in water were performed toremove additional residual polymer. Scanning electron micrographsrevealed the formation of sub-micron sized microspheres often arrangedin a string-like structure.

Example 8 Preparation of Bovine Serum Albumin Microspheres UsingEudragit Registered TM E100

[0248] Bovine serum albumin microspheres were prepared using Eudragit®E110 polymer. This polymer is soluble in an organic solvent that ismiscible with water.

[0249] Eudragit® E100 polymer (Rohm, Malden, Mass.) was dissolved in a1:1 solution of 0.1M sodium acetate buffer (pH 5.0) and ethanol. Thefinal pH of the solution was pH 6.5. A 400 ml aliquot of a 10 mg/mlsolution of bovine serum albumin (BSA, Sigma Chemical Co., St. Louis,Mo.) was combined with 800 ml of the Eudragit® E100 polymer solution. Aclear solution formed. The BSA/polymer solution was incubated in a 70°C. water bath for 30 minutes. A milky white suspension was observed,indicating microsphere formation. The particles were collected bycentrifugation for 10 minutes at 8,000 rpm and decantation of theliquid.

Example 9 Preparation of Insulin Microspheres Using Dextran

[0250] Insulin microspheres were prepared using dextran polymer.

[0251] A 20% (weight/weight) solution of dextran polymer (MW 500,000,Sigma Chemical Co., St. Louis, Mo.) was dissolved in pH 5.0 sodiumacetate buffer. 1.9 ml of dH2O was added to 20.5 mg of insulin (SigmaChemical Co.). 100 ml of 0.2M HCL was added to dissolve the insulin. A400 ml aliquot of the insulin solution was placed in a test tube. An 800ml aliquot of the dextran solution was added to the insulin solution.The mixture was vortexed. The mixture turned cloudy upon addition of thedextran solution. The tubes were heated to a final temperature of either70° C. or 90° C. to form insulin microspheres. The microspheres werecentrifuged at 10,000 rpm for 5 minutes and the liquid decanted toremove residual polymer. The microspheres were washed with 10% ethanolin water.

[0252] The microspheres were place in a phosphate buffered salinesolution, pH 7.4 to determine the dissolution characteristics. Theinsulin particles formed at a final temperature of 90° C. did notdissolve in the phosphate buffered saline whereas the insulin particlesprepared at a final temperature of 70° C. dissolved within 15 minutes.Therefore, insulin particle stability may be adjusted by varying theincubation temperature employed during particle formation.

Example 10 Preparation of Microspheres Using Various Polymers

[0253] Human serum albumin microspheres were prepared using ninedifferent polymers or mixtures of polymers.

[0254] Procedure: A protein solution (1-5% human serum albumin, pH4.5-5.5 or bovine serum albumin) in a buffer was prepared. The followingpolymer solutions were prepared:

[0255] polyethylene glycol/polyvinylpyrrolidone (3 kDa PEG/40 kDa PEG ina 1:1 mixture); hetastarch (500 kDa); Pluronic L-101™ polymer; dextran(3-500 kDa); dextran sulfate (3-500 kDa); polyvinylpyrrolidone (10-360kDa); polyethylene glycol/polyvinylpyrrolidone with inulin (apolysaccharide); polyethylene glycol/polyvinylpyrrolidone with PluronicL-101™ polymer; dextran with Pluronic L-101™ polymer. Approximately onevolume of protein solution was mixed with two volumes of polymersolution. The mixture was incubated in a water bath at 70° C. to 90° C.for thirty minutes. The mixture was then placed in an ice bath.Microsphere formation was observed.

[0256] The microspheres were centrifuged until compacted into a pellet,the supernatant decanted, and the pellet washed twice in a 10% ethanolin water solution to remove the residual polymer solution. Microsphereswere then washed three times with deionized water. The microspheres wereused or tested immediately or were lyophilized for subsequent use.

[0257] Observations: Microspheres prepared using polymers having ahigher molecular weight or a higher concentration of polymers provide amore viscous medium which produced a more uniform microsphere sizedistribution. The inclusion of a surfactant, such as Pluronic L-101™polymer or mixing during microsphere formation affected microspheresize. An increase in protein concentration during microsphere formationcaused an increase in the incorporation of protein into themicrospheres. An increase in polymer size generally caused an increasein protein incorporation into the microspheres. The use of differentpolymers affected release kinetics. For example bovine serum albumin(BSA) microspheres prepared using dextran released approximately 15%less BSA than microspheres prepared using PEG/PVP. However, the releaseof polysaccharide from protein-polysaccharide microspheres, such as therelease of [3] H-inulin from human serum albumin/inulin microspheres,was more rapid when dextran rather than PEG/PVP was employed.

Example 11 Preparation of Protein Microspheres Containing NafarelinAcetate Using Various Polymers

[0258] Protein microspheres containing the drug nafarelin acetate wereprepared.

[0259] Nafarelin acetate is useful in the treatment of endometriosis,precocious puberty and prostate cancer.

[0260] Procedure: Human serum albumin microspheres were prepared asdescribed above in Example 10 using nine different polymers or mixturesof polymers. Nafarelin acetate (Roche Laboratories, Nutley, N.J.) wasdissolved in deionized water to produce a 10 mg/ml solution.

[0261] To 1 mg of nafarelin acetate was added 10 mg of the human serumalbumin microspheres. The mixture was vortexed and mixed for 16 hours at4° C. A 3M ammonium sulfate solution was added to the mixture to a finalconcentration of 0.5M and vortexed and mixed for 15 minutes at ambienttemperature. A 1M zinc sulfate solution was added to a finalconcentration of either 0.01 M, 0.1 M or 1 M and vortexed and mixed for1 hour at ambient temperature.

[0262] The mixtures were centrifuged, supernatant decanted and pelletresuspended in deionized water three times to wash the microspheres.

[0263] Observations: The addition of zinc sulfate reduced the rate ofrelease of nafarelin acetate from human serum albumin microspheres. Theresults are shown in FIG. 9.

Example 12 Preparation of Doxorubicin/Albumin Microspheres

[0264] Human serum albumin microspheres containing the chemotherapeuticdrug doxorubicin were prepared.

[0265] To 1 mL of a solution of 250 mg/mL human serum albumin (Sigma,St. Louis, Mo.) in dH2O was added 0.05 mL of a 5 mg/mL solution ofdoxorubicin (Sigma Chemical Co., St. Louis, Mo.) in dH2O, the combinedsolution was mixed and allowed to stand at room temperature for 30minutes. To the above solution was added 3.0 mL of a solution of 200mg/mL dextran sulfate (MW 500,000, Sigma Chemical Co., St. Louis, Mo.)in dH2O, and the resulting solution was mixed and incubated at 37° C.for 30 minutes. The solution was then incubated at 70° C. for 30minutes, after which 1.0 mL of 3.0M sodium acetate, pH 5.0, was addedand the resulting solution mixed. The solution was then incubated at 90°C. for 30 minutes. Microspheres were formed. The microspheres werewashed two times with 5.0 mL dH2O.

Example 13 Incorporation of LHRH into Dextran Sulfate/AlbuminMicrospheres

[0266] Luteinizing hormone releasing hormone was incorporated intomicrospheres composed of human serum albumin and dextran sulfatemicrospheres.

[0267] To 0.168 mL of a 10% (weight/volume) solution of dextran sulfate(average MW 500,000, Sigma Chemical Co., St. Louis, Mo.) in dH2O wasadded 0.25 mL of a 20% (weight/volume) solution of human serum albumin(Sigma Chemical Company, St. Louis, Mo.) in dH2O. The solution wasthoroughly mixed, and 0.856 mL of a solution of 25% (weight/volume)polyethylene glycol (average MW 3.35 kDa, Spectrum, Gardena, Calif.) and25% (weight/volume)polyvinylpyrrolidone (average MW 40 kDa, Spectrum,Gardena, Calif.) in an aqueous solution of 0.1M sodium acetate, pH 5.5was added.

[0268] The resulting solution was thoroughly mixed and placed in a waterbath at a temperature between 70° C. and 90° C. for between 30 minutesand 3 hours. Microspheres were formed. Microspheres were collected bycentrifugation at 17.5K×g for 10 minutes, resuspended for washing in 0.5mL of dH2O, and collected again by centrifugation. The microspheres wereresuspended in 0.3 mL of a solution of 0.1M sodium acetate, pH 5.5. A0.01 mL aliquot of a 10 mg/mL solution of luteinizing hormone releasinghormone (LHRH, Sigma Chemical Co., St. Louis, Mo.) in dH2O was added andincubated for 16 hours at room temperature to allow the LHRH peptide tobind to the microspheres. After the 16 hour binding incubation, theunbound LHRH peptide was removed by two cycles of washes with dH2O andcollection by centrifugation. The yield of LHRH incorporation wastypically 83 to 90%.

Example 14 Incorporation of estradiol into ProMaxx® Microspheres

[0269] Human serum albumin microspheres were formed by combining 12.5 wt% polyethylene glycol and 12.5 wt % polyvinyl pyrollidone in 50 mMsodium acetate buffer at pH 5.3 and with 1% (wt/vol) human serum albumindissolved in aqueous solution. The solution was placed at 70° C. for 30minutes. The microspheres were formed and precipitated from solution.The microsphere suspension was washed 3 times in deionized water.Estradiol was then loaded into human serum albumin microspheres at 3different weight percent loadings. The estradiol was dissolved in ethylalcohol and incubated in the presence of the albumin microspheres. Theaffinity of estradiol for albumin by means of hydrophobic interactionsresulted in the ability to load estradiol at various weight percentloadings. The 10% by weight estradiol containing microspheres contained1 mg of estradiol and 8.33 mg of Human Serum Albumin (HSA). The 49% byweight estradiol containing microspheres contained 8.15 mg of estradioland 8.33 mg of HSA. The 77% by weight estradiol containing microspherescontained 27.68 mg of estradiol and 8.33 mg of HSA The microspheres werereleased into phosphate buffered saline (PBS) at pH 7.4.

[0270] All samples were rotated at 20 rpm at 37° C. to insure constantmixing. One (1) mL of PBS was added to the microspheres which werehoused in standard polypropylene centrifuge tubes. At each time point,the tubes were centrifuged, the supernatant removed, and the 1 mL ofrelease media was placed in a sample vial. Each 1 mL sample was speedvacuum dried and resuspended in 200 μL of ethanol. The releasedestradiol was measured by HPLC.

[0271] The sustained release of estradiol in shown in FIG. 10.

[0272] The sustained release of estradiol was also demonstrated in vivo.The microspheres were injected into five dogs and the resulting serumconcentrations were determined by radioimmuno assay for estradiol. Theresults are shown in FIG. 11. The three groups consisting of five dogseach included an estradiol control which resulted in the lowestconcentrations and the shortest half life in vivo. ProMaxx® which arethe albumin microspheres loaded with estradiol yielded substantiallygreater plasma estradiol concentrations with a significantly longer halflife. And when the estradiol-microspheres were chemically stabilizedwith EDC (Ethyl dimethyl amino propyl carbodiimide), greater sustainedplasma levels and increased half life of estradiol was observed over a43 day period.

Example 15 Incorporation of diclofenac Na into ProMaxx® Microspheres

[0273] Bovine serum albumin microspheres were formed by combining 12.5wt % polyethylene glycol and 12.5 wt % polyvinyl pyrollidone in 50 mMsodium acetate buffer at pH 5.3 and with bovine serum albumin dissolvedin aqueous solution. The solution was placed at 70° C. for 30 minutes.The microspheres formed and precipitated from solution. The microspheresuspension was washed 3 times in deionized water.

[0274] The non steroidal anti inflammatory drug diclofenac Na was boundto albumin microspheres by manipulating the degree of ionization ofdiclofenac Na by altering the pH of the incubating solutions. Theefficiency of diclofenac Na incorporation could be optimized to 65%incorporation by adjusting the incubating conditions to a pH of as shownin FIG. 12.

Example 16 Formation of Leuprolide Acetate-Containing Microspheres

[0275] Leuprolide acetate is an analog of luteinizing hormone releasinghormone.

[0276] An aqueous solution of albumin (e.g., human, bovine, mouse, orrat serum albumin) is added to an aqueous solution of anionic complexingagent (dextran sulfate of molecular weight (MW) 10 kDa to 500 kDa orheparin or heparan sulfate or polyglutamic or polyaspartic acids) withor without an aqueous solution of divalent cations (calcium or magnesiumor zinc) at ambient temperature and the resulting mixture is agitatedfor a period of time from 5 minutes to 3 hours. To this mixture is thenadded an aqueous solution of polymer (either polyethylene glycol (PEG)of MW 3.5 to 40 kDa; polyvinylpyrrolidone (PVP) of MW 6 to 40 kDa, ormixtures of both PEG and PVP, or starch or hydroxyethylstarch of MW 500kDa). The concentration of albumin in the final mixture is from 5-30g/L, of ionic complexing agent is from 5-30 g/L, of divalent cationsfrom 0-30 g/L, and polymer concentration is from 100 μL to 400 g/L. Themixture is agitated for a period from 5 minutes to 6 hours at ambienttemperature. The temperature of the mixture is then raised to atemperature between 37° C. and 50° C. over a period of from 5 minutes to3 hours. The microspheres are subjected to stabilization by the additionof a chemical stabilizer (EDC, or other linking agent) or by thermalstabilization by heating to a temperature of between 70° C. and 100° C.over a period of 0.5 to 6 hours. The mixture is subjected to stabilizerfor a period of from 10 minutes to 16 hours or for thermal stabilizationthe temperature is maintained at the final value from 10 minutes to 6hours. The microspheres are then collected and separated from theunincorporated components of the reaction mixture by filtration orcentrifugation with subsequent washing or by diafiltration.Incorporation of leuprolide acetate to the microspheres is effected byadding an aqueous solution of leuprolide solution to an aqueoussuspension of microspheres. The concentrations of microspheres are 1-50g/L of leuprolide acetate are 1-100 g/L in an aqueous medium of from0-100 mM buffer at pH between 2.5 and 10.0. After incubation of theleuprolide acetate with the microsphere suspension for 5 minutes to 8hours at 5-50° C., peptide-containing microspheres are washed by themethods described above to remove unbound peptide. Thepeptide-containing microspheres are in some cases subjected to furtherstabilization by a repeat exposure to chemical stabilizers as describedabove, and then washed to remove unbound peptide. The finalpeptide-containing microspheres are then either resuspended in water orlypophylized. Their pharmaceutic efficacy is determined using apharmacodynamic rat model of testosterone suppression over periods from7 to 180 days. Leuprolide acetate-containing microspheres fabricatedaccording to the above method have been injected into rats andleuprolide acetate has been detected in serum for from 7 to 120 days.Rat serum testosterone has been suppressed in rats so injected with theleuprolide acetate-containing microspheres for from 7 to 120 days. Theseresults evidence the sustained release of physiologically activeleuprolide acetate over this time period.

Example 17 Examples of Calcium-Containing ProMaxx Microspheres

[0277] The following example illustrates specific conditions for formingpreferred microspheres of the invention. It is to be understood that theconcentrations, times, and pH's can vary within a reasonableexperimental amount and still result in microspheres having thecharacteristics described below. As described in the detaileddescription of the invention, it is preferred that the divalent metal(preferably calcium or magnesium) be added during the formation processand that such addition of the metal occur within 30 minutes of theaddition of the other components which are used for formation of themicrospheres. Further details regarding the microsphere formationprocedure are provided below.

[0278] To 1 volume of a 25% (w/v) solution of dextran sulfate (avg MW500 kDa) is added approximately 2 volumes of a 25% (w/v) solution ofalbumin (human) and the mixture is stirred thoroughly for at least 5min. To this mixture approximately 6 volumes of a 36% (weight/volume)solution of hetastarch in aqueous 63 mM Na acetate is stirred thoroughlyand to this hetastarch solution is added water while continuing thoroughstirring. The resulting solution is stirred thoroughly for at least 5min. whereupon the dextran sulfate-albumin mixture is added slowly toit. To this mixture a suitable volume of a 1.5 M solution of CaCl₂,ZnCl₂, or MgCl₂ is added to make a final mixture containing 25 mM or 100mM metal cation. This mixture is constantly thoroughly stirred. Thetemperature of the reaction mixture is then increased to a finaltemperature of 60-90° C. over a period of 50-60 min. The temperature ofthe mixture is maintained at 60-90° C. for 30-120 min., whereupon anequal volume of a room temperature 25 mM solution of MES-Na⁺(N-morpholinoethanesulfonic acid) at pH 6.0±0.5 is added while stirringis continued. The formed microspheres are then diafiltered against 1vol. Of 25 mM MES pH 6.0±0.5. The microspheres are then concentrated anddiafiltered against 9 volumes of 25 mM MES pH 6.0±0.5 at roomtemperature. The loading and diafiltration are carried out at pH 4.5±0.5in the case of Zn²⁺-containing microspheres. The microspheres areconcentrated by diafiltration. To the microsphere suspension containing17.1 g of albumin (human) is added a solution of 3.43 g leuprolideacetate in 25 mM MES pH 6.0 at a rate of 0.4 liters per min. Theleuprolide solution addition is followed by addition of MES solution tothe leuprolide acetate-microsphere suspension. The suspension is mixedfor 15 min. at room temperature, whereupon the suspension isconcentrated by diafiltration. To this suspension is added a solution of17.14 g EDC ((3,3-ethyl-dimethylaminopropyl) carbodiimide) in 25 mM MESpH 6.0±0.5 at 0.4 liters per min. The suspension is incubated at roomtemperature while stirring for 180 min., following which it isdiafiltered against 10 vol. Of distilled water and concentrated bydiafiltration. The leuprolide acetate content of the suspension isdetermined whereupon a 50% (w/v) solution of sucrose in water is addedand diluted such that the final suspension is approximately 3.75-4.025mg/mL leuprolide acetate in 5% (w/v) sucrose. This bulk suspension isthen filled at 2.0 mL per vial and lyophilized.

[0279] (1) Physical Characteristics

[0280] Loading characteristics—The Mg²⁺- and Ca²⁺-containingmicrospheres bound the drug in high yield (>90%), while theZn²⁺-containing microspheres bound the drug at less than 70% yield.

[0281] (2) In Vitro Release

[0282] The Mg²⁺- and Ca²⁺-containing microspheres had similar releaserates of leuprolide that did not vary with the concentration of metalcation present during microsphere fabrication. At 25 mM Zn²⁺, therelease rate was similar to that of the Ca²⁺ and Mg²⁺-containingmicrospheres, but at 100 mM Zn²⁺, the magnitude of the initial “burst”phase was greater.

[0283] (3) In Vivo Performance

[0284] In rats, at identical dosage of drug, the Ca²⁺- andMg²⁺-containing microspheres suppressed serum testosterone to castratelevels for at least 4 weeks. The Zn²⁺-containing microspheres did notsuppress testosterone for even three weeks in the rat model.

[0285] It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit and scope ofthe invention. It is intended to encompass all such modifications withinthe scope of the appended claims.

[0286] All references, patents and patent publications that are recitedin this application are incorporated in their entirety herein byreference.

What is claimed is:
 1. A microsphere comprising: (1) a macromolecule;(2) a water soluble polymer; (3) a polyanionic first complexing agent;and (4) a divalent metal cation second complexing agent selected fromthe group consisting of calcium and magnesium.
 2. The microsphere ofclaim 1, wherein the macromolecule is a protein.
 3. The microsphere ofclaim 2, wherein the protein is selected from the group consisting of:Albumins (preferably, human serum albumin), HAS; BSA, IgG, IgM, insulin,hGH, lysozyme, alpha-lactoglobulin, basic fibroblast growth factor,VEGF, chymotrypsin, trypsin, carbonic anhydrase, ovalbumin,phosphorylase b, alkaline phosphatase, beta-galactosidase, fibrinogen,poly-1-lysine, immunoglobulins (e.g., antibodies), casein, collagen, soyprotein, gelatin, insulin, human growth hormone, GCSF, GMCSF, LHRH,VEGF, basic fibroblast growth factor (bFGF), asparaginase, tPA,urokinase, streptokinase, interferon, glucagon, ACTH, oxytocin,secretin, vasopressin, and levothyroxin.
 4. The microsphere of claim 3,wherein the protein is an albumin.
 5. The microsphere of claim 3,wherein the protein is an immunoglobulin.
 6. The microsphere of any ofclaims 2-5, wherein the microsphere contains from 40 to less than 100%protein.
 7. The microsphere of any of the preceding claims, wherein thewater soluble polymer is selected from the group consisting of:carbohydrate-based polymers, such as methylcellulose, carboxymethylcellulose-based polymers, dextran, polydextrose, derivatized chitins,chitosan, starch (including hetastarch) and derivatives thereof,polyaliphatic alcohols, such as polyethylene oxide and derivativesthereof including polyethylene glycol (PEG), PEG-acrylates, polyethyleneimine, polyvinyl acetate, and derivatives thereof, poly(vinyl) polymers,such as poly(vinyl) alcohol, poly(vinyl)pyrrolidone,poly(vinyl)phosphate, poly(vinyl)phosphonic acid and derivativesthereof, polyacrylic acids and derivatives thereof, polyorganic acids,such as polymaleic acid, and derivatives thereof, polyamino acids, suchas polylysine and polyimino acids, such as polyimino tyrosine andderivatives thereof, co-polymers and block co-polymers, such aspoloxamer 407 or Pluronic L-101 TM polymer and derivatives thereof,tert-polymers and derivatives thereof, polyethers, such aspoly(tetramethylene ether glycol) and derivatives thereof, naturallyoccurring polymers, such as zein and pullulan and derivatives thereof,polyimids, such as polyn-tris(hydroxymethyl)methylmethacrylate andderivatives thereof, surfactants, such as polyoxyethylene sorbitan andderivatives thereof, polyesters such as poly(ethyleneglycol)(n)monomethyl ether mono(succinimidylsuccinate)ester andderivatives thereof, branched and cyclo-polymers, such as branched PEGand cyclodextrins and derivatives thereof, and polyaldehydes, such aspoly(perfluoropropylene oxide-b-perfluoroformaldehyde) and derivativesthereof.
 8. The microsphere of claim 7, wherein the water solublepolymer is a carbohydrate-based polymer.
 9. The microsphere of claim 7,wherein the starch is a hydroxyethylstarch.
 10. The microsphere of claim7, wherein the starch is a hetastarch.
 11. The microsphere of claim 1,wherein the polyanionic first complexing agent is a polyanionicpolysaccharide.
 12. The microsphere of claim 11, wherein the polyanionicpolysaccharide is selected from the group consisting of: dextransulfate, galacturonic acids, alginates, mannuronic acid, guluronic acid,hyaluronic acid, chondroitin sulfates, heparin, chitin, chitosan,glycosaminoglycans, and proteoglycans.
 13. The microsphere of claim 12,wherein the polyanionic polysaccharide is dextran sulfate.
 14. Themicrosphere of claim 1, wherein the divalent metal cation is calcium.15. The microsphere of claim 1, wherein the divalent metal cation ismagnesium.
 16. The microsphere of claim 1, wherein the microsphere has asmooth surface that includes a plurality of channel openings, each ofsaid channel openings having a diameter that is less than 1000Angstroms.
 17. The microsphere of claim 1, wherein the microsphere doesnot contain detectable oil or organic solvent.
 18. The microsphere ofclaim 1, wherein the macromolecule is albumin, and the water solublepolymer is a carbohydrate-based polymer.
 19. The microsphere of claim18, wherein the albumin is human serum albumin, and wherein thecarbohydrate based polymer is hetastarch.
 20. The microsphere of claim1, further comprising an active agent.
 21. The microsphere of claim 20,wherein the active agent is a therapeutic agent.
 22. The microsphere ofclaim 20, wherein the active agent is a diagnostic agent.
 23. Themicrosphere of claim 20 wherein, the active agent is selected from thegroup consisting of: a hormone, an antibiotic, an antiinfective agent, ahematopoietic, a thrombopoietic agent, an antidementia agent, anantiviral agent, an antitumoral agent, an antipyretic, an analgesic, anantiinflammatory agent, an antiulcer agent, an antiallergic agent, anantidepressant, a psychotropic agent, a cardiotonic, an antiarrythmicagent, a vasodilator, an antihypertensive agent, an antidiabetic agent,an anticoagulant, a cholesterol lowering agent, a therapeutic agent forosteoporosis, an enzyme, a vaccine, an immunological agent, an adjuvant,a cytokine, a growth factor, a nucleotide, a nucleic acid, acarbohydrate, a polysaccharide, a virus, and a virus particle.
 24. Themethod of claim 20, wherein the active agent is a luteinizing hormonereleasing hormone or analog thereof.
 25. The method of claim 20, whereinthe active agent is leuprolide.
 26. The microsphere of claim 20, whereinthe first polyanionic complexing agent is dextran sulfate and the activeagent is leuprolide acetate.
 27. A syringe containing a single dose ofthe microspheres of any of the preceding claims, wherein the syringepreferably further includes a needle having a bore size that is from 14to 30 gauge.
 28. A microsphere comprising: (1) a macromolecule; (2) awater soluble polymer; and (3) a polycationic complexing agent.
 29. Themicrosphere of claim 28, wherein the macromolecule is a nucleic acid.30. The microsphere of claim 29, wherein the nucleic acid is selectedfrom the group consisting of: DNA, RNA, plasmid, viral vector,oligonucleotide, antisense nucleic acids, and missense nucleic acds. 31.The microsphere of any of claims 28-30, wherein the microsphere containsfrom 40 to less than 100% macromolecule.
 32. The microsphere of any ofclaims 28-31, wherein the water soluble polymer is selected from thegroup consisting of: carbohydrate-based polymers, such asmethylcellulose, carboxymethyl cellulose-based polymers, dextran,polydextrose, derivatized chitins, chitosan, and starch (includinghetastarch) and derivatives thereof, polyaliphatic alcohols, such aspolyethylene oxide and derivatives thereof including polyethylene glycol(PEG), PEG-acrylates, polyethylene imine, polyvinyl acetate andderivatives thereof, poly(vinyl) polymers, such as poly(vinyl) alcohol,poly(vinyl)pyrrolidone, poly(vinyl)phosphate, poly(vinyl)phosphonic acidand derivatives thereof, polyacrylic acids and derivatives thereof,polyorganic acids, such as polymaleic acid and derivatives thereof,polyamino acids, such as polylysine and polyimino acids, such aspolyimino tyrosine and derivatives thereof, co-polymers and blockco-polymers, such as poloxamer 407 or Pluronic L-101 TM polymer andderivatives thereof, tert-polymers and derivatives thereof, polyethers,such as poly(tetramethylene ether glycol) and derivatives thereof,naturally occurring polymers, such as zein and pullulan and derivativesthereof, polyimids, such as polyn-tris(hydroxymethyl)methylmethacrylateand derivatives thereof, surfactants, such as polyoxyethylene sorbitanand derivatives thereof, polyesters, such as poly(ethyleneglycol)(n)monomethyl ether mono(succinimidylsuccinate)ester andderivatives thereof, branched and cyclo-polymers, such as branched PEGand cyclodextrins and derivatives thereof and polyaldehydes, such aspoly(perfluoropropylene oxide-b-perfluoroformaldehyde) and derivativesthereof.
 33. The microsphere of claim 32, wherein the water solublepolymer is a carbohydrate-based polymer.
 34. The microsphere of claim32, wherein the starch is a hydroxyethylstarch.
 35. The microsphere ofclaim 32, wherein the starch is a hetastarch.
 36. The microsphere ofclaims 28-35, wherein the polycationic complexing agent is selected fromthe group consisting of: poly-lysine, poly-arginine, poly-imino acids,poly-imino tyrosine, cholestyramine-resin, diethyl amino ethylcellulose, poly-citrulline, and poly-ornithine.
 37. The microsphere ofclaim 36, wherein the polycationic complexing agent is polylysine. 38.The microsphere of claims 28-37, wherein the microsphere has a smoothsurface that includes a plurality of channel openings, each of saidchannel openings having a diameter that is less than 1000 Angstroms. 39.The microsphere of claims 28-38, wherein the microsphere does notcontain detectable oil or organic solvent.
 40. The microsphere of claim28, further comprising an active agent.
 41. The microsphere of claim 40,wherein the active agent is a therapeutic agent.
 42. The microsphere ofclaim 40, wherein the active agent is a diagnostic agent.
 43. Themicrosphere of claim 40 wherein, the active agent is selected from thegroup consisting of: a hormone, an antibiotic, an antiinfective agent, ahematopoietic, a thrombopoietic agent, an antidementia agent, anantiviral agent, an antitumoral agent, an antipyretic, an analgesic, anantiinflammatory agent, an antiulcer agent, an antiallergic agent, anantidepressant, a psychotropic agent, a cardiotonic, an antiarrythmicagent, a vasodilator, an antihypertensive agent, an antidiabetic agent,an anticoagulant, a cholesterol lowering agent, a therapeutic agent forosteoporosis, an enzyme, a vaccine, an immunological agent, an adjuvant,a cytokine, a growth factor, a nucleotide, a nucleic acid, acarbohydrate, a polysaccharide, a virus, and a virus particle.
 44. Themethod of claim 40, wherein the active agent is a luteinizing hormonereleasing hormone or analog thereof.
 45. The method of claim 40, whereinthe active agent is leuprolide.
 46. A syringe containing a single doseof the microspheres of any of claims 28-45, wherein the syringepreferably further includes a needle having a bore size that is from 14to 30 gauge.
 47. A method for forming a microsphere comprising: (1)forming an aqueous mixture containing: (a) a macromolecule; (b) a watersoluble polymer; and (c) a polycationic complexing agent; (2) allowingthe microspheres to form in the aqueous mixture; and (3) stabilizing themicrospheres, preferably by contacting the microspheres with acrosslinking agent and/or exposing the microspheres to an energy source,preferably heat, under conditions sufficient to stabilize themicrospheres.
 48. The method of claim 47, wherein forming the aqueousmixture is performed by combining the macromolecule, the water solublepolymer, and the polycationic complexing agent essentiallysimultaneously.
 49. The method of claim 47, wherein the macromolecule isa nucleic acid.
 50. The method of claim 49, wherein the nucleic acid isselected from the group consisting of: DNA, RNA, plasmid, viral vector,oligonucleotide, antisense nucleic acids, and missense nucleic acids.51. The method of claim 47, further comprising the step of: (4)contacting the microsphere with a solution of an active agent, toincorporate the active agent into the microsphere.
 52. The method ofclaim 51, wherein the active agent is a therapeutic agent.
 53. Themethod of claim 51, wherein the active agent is a diagnostic agent. 54.The method of claim 51, wherein the step of contacting the microspherewith the solution of active agent results in a yield of incorporation ofat least 60% of the active agent.
 55. The method of claim 51, whereinthe active agent is selected from the group consisting of: a hormone, anantibiotic, an antiinfective agent, a hematopoietic, a thrombopoieticagent, an antidementia agent, an antiviral agent, an antitumoral agent,an antipyretic, an analgesic, an antiinflammatory agent, an antiulceragent, an antiallergic agent, an antidepressant, a psychotropic agent, acardiotonic, an antiarrythmic agent, a vasodilator, an antihypertensiveagent, an antidiabetic agent, an anticoagulant, a cholesterol loweringagent, a therapeutic agent for osteoporosis, an enzyme, a vaccine, animmunological agent, an adjuvant, a cytokine, a growth factor, anucleotide, a nucleic acid, a carbohydrate, a polysaccharide, a virus,and a virus particle.
 56. The method of claim 51, wherein the activeagent is a luteinizing hormone releasing hormone or analog thereof. 57.The method of claim 51, wherein the active agent is leuprolide.
 58. Themethod of claim 51, wherein the step of contacting the microsphere withthe solution of an active agent results in a yield of incorporation ofat least 70% of the active agent.
 59. The method of claim 51, whereinthe step of contacting the microsphere with the solution of an activeagent results in a yield of incorporation of at least 80% of the activeagent.
 60. The method of claim 51, wherein the step of contacting themicrosphere with the solution of an active agent results in a yield ofincorporation of at least 90% of the active agent.
 61. The method ofclaim 51, wherein the step of contacting the microsphere with thesolution of an active agent results in a yield of incorporation of atleast 95% of the active agent.
 62. The method of claim 51, wherein thestep of contacting the microsphere with the solution of an active agentresults in a yield of incorporation of at least 98% of the active agent.63. The method of claim 47, wherein the step of stabilizing comprisesexposing the microspheres to an energy source.
 64. The method of claim63, wherein the energy source is heat.
 65. The method of claim 47,wherein the step of stabilizing comprises contacting the microsphereswith a crosslinking agent.